Toxicity Management for Anti-Tumor Activity of CARs

ABSTRACT

The present invention provides compositions and methods for treating cancer in a patient. In one embodiment, the method comprises a first-line therapy comprising administering to a patient in need thereof a genetically modified T cell expressing a CAR wherein the CAR comprises an antigen binding domain, a transmembrane domain, a costimulatory signaling region, and a CD3 zeta signaling domain and monitoring the levels of cytokines in the patient post T cell infusion to determine the type of second-line of therapy appropriate for treating the patient as a consequence of the presence of the CAR T cell in the patient.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 14/410,659, filed Dec. 23, 2014, which is a U.S. national phaseapplication filed under 35 U.S.C. § 371 claiming benefit toInternational Patent Application No. PCT/US2013/050267 filed on Jul. 12,2013, which is entitled to priority under 35 U.S.C. § 119(e) to U.S.Provisional Application No. 61/671,482, filed Jul. 13, 2012 and U.S.Provisional Application No. 61/782,982, filed Mar. 14, 2013, each ofwhich is hereby incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

Patients with relapsed and chemotherapy-refractory acute lymphocyticleukemia (ALL) have a poor prognosis despite the use of aggressivetherapies such as allogeneic hematopoietic stem cell transplantation(Barrett et al., 1994, N Engl J Med 331:1253-8; Gokbuget et al., 2012,Blood 120:2032-41) and bi-specific CD19 antibody fragments (Bargou etal., 2008, Science 321:974-7). Chimeric antigen receptor modified Tcells targeting lineage-specific antigens CD19 and CD20 have beenreported to be effective in adults with CLL and B-cell lymphomas (Tillet al., 2008, Blood 112:2261-71; Kochenderfer et al., 2010, Blood116:4099-102; Brentjens et al., 2011, Blood 118:4817-28; Porter et al.,2011, N Engl J Med 365:725-33; Kalos et al., 2011, Science TranslationalMedicine 3:95ra73; Savoldo et al., 2011, J Clin Invest 121:1822-5).However, the effects of CAR T cells on ALL blasts, a more immatureleukemia with a more rapid progression, have not been fullyinvestigated.

Delayed onset of the tumor lysis syndrome and cytokine secretion,combined with vigorous in vivo chimeric antigen receptor T-cellexpansion has been reported (Porter et al., 2011, N Engl J Med365:725-33; Kalos et al., 2011, Science Translational Medicine3:95ra73). However, the effects of cytokine secretion and disordersassociated with in vivo chimeric antigen recept T-cell expansion havenot been fully investigated.

Thus, there is an urgent need in the art for compositions and methodsfor treatment of cancer using CARs and addressing toxicity of the CARs.The present invention addresses this need.

SUMMARY OF THE INVENTION

The invention provides a method of treating a patient having a disease,disorder or condition associated with an elevated expression of a tumorantigen. In one embodiment, the method comprises administering afirst-line therapy and a second-line therapy to a patient in needthereof, wherein the first line therapy comprises administering to thepatient an effective amount of a cell genetically modified to express aCAR, wherein the CAR comprises an antigen binding domain, atransmembrane domain, and an intracellular signaling domain.

In one embodiment, following the administration of the first-linetherapy, cytokine levels in the patient are monitored to determine theappropriate type of second-line therapy to be administered to thepatient and the appropriate second-line therapy is administered to thepatient in need thereof.

In one embodiment, an increase in the level of a cytokine identifies atype of cytokine inhibitory therapy to be administered to the patient inneed thereof.

In one embodiment, the cytokine is selected from the group consisting ofIL-10, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, IL-13, IL-15, IL-17,IL-1Rα, IL-2R, IFN-α, IFN-γ, MIP-1α, MCP-1β, TNFα, GM-CSF, G-CSF, CXCL9,CXCL10, CXCR factors, VEGF, RANTES, EOTAXIN, EGF, HGF, FGF-0, CD40,CD40L, ferritin, and any combination thereof.

In one embodiment, the cytokine inhibitory therapy is selected from thegroup consisting of a small interfering RNA (siRNA), a microRNA, anantisense nucleic acid, a ribozyme, an expression vector encoding atransdominant negative mutant, an antibody, a peptide, a small molecule,a cytokine inhibitory drug, and any combination thereof.

In one embodiment, the cytokine levels are monitored by detecting theprotein level of the cytokine in a biological sample from the patient.

In one embodiment, the cytokine levels are monitored by detecting thenucleic acid level of the cytokine in a biological sample from thepatient.

The invention provides a method of reducing or avoiding an adverseeffect associated with the administration of a cell genetically modifiedto express a CAR, wherein the CAR comprises an antigen binding domain, atransmembrane domain, and an intracellular signaling domain, the methodcomprising monitoring the levels of a cytokine in a patient to determinethe appropriate type of cytokine therapy to be administered to thepatient and administering the appropriate cytokine therapy to thepatient.

In one embodiment, an increase in the level of a cytokine identifies atype of cytokine inhibitory therapy to be administered to the patient.

In one embodiment, the cytokine is selected from the group consisting ofIL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, IL-13, IL-15, IL-17,IL-1Rα, IL-2R, IFN-α, IFN-γ, MIP-1α, MIP-1β, MCP-1, TNFα, GM-CSF, G-CSF,CXCL9, CXCL10, CXCR factors, VEGF, RANTES, EOTAXIN, EGF, HGF, FGF-β,CD40, CD40L, ferritin, and any combination thereof.

In one embodiment, the cytokine inhibitory therapy is selected from thegroup consisting of a small interfering RNA (siRNA), a microRNA, anantisense nucleic acid, a ribozyme, an expression vector encoding atransdominant negative mutant, an intracellular antibody, a peptide, asmall molecule, a cytokine inhibitory drug, and any combination thereof.

In one embodiment, the cytokine levels are monitored by detecting theprotein level of the cytokine in a biological sample from the patient.

In one embodiment, the cytokine levels are monitored by detecting thenucleic acid level of the cytokine in a biological sample from thepatient.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description of preferred embodiments of theinvention will be better understood when read in conjunction with theappended drawings. For the purpose of illustrating the invention, thereare shown in the drawings embodiments which are presently preferred. Itshould be understood, however, that the invention is not limited to theprecise arrangements and instrumentalities of the embodiments shown inthe drawings.

FIG. 1 is an image demonstrating serum cytokine levels in four differentpatients. All patients exhibited cytokine release, including IL-6.

FIG. 2 is an image depicting serum cytokines plotted in a representativepatient. The patient was critically ill on days 5 to 7, and only beganto improve following tocilizumab administration.

FIG. 3 is an image demonstrating that antibody interventions do notimpact CART 19 cellular functionality as measured for markers of T cellactivity (perforin and IFN-γ).

FIG. 4, comprising FIGS. 4A through 4C, is a series of images depictingclinical responses. FIG. 4A shows two children with multiply-relapsedchemotherapy-refractory CD19+B cell precursor acute lymphoblasticleukemia who were treated with CTL019 cells, infused on Day 0. Changesin serum lactate dehydrogenase (LDH) and body temperature after CTL019infusion, with maximum temperature per 24 hour period demarcated withcircles. CHOP-100 was given methylprednisolone starting on day 5 at 2mg/kg/day, tapered to off by day 12. On the morning of day 7, etanerceptwas given 0.8 mg/kg×1. At 6 μm in the evening of day 7, tocilizumab 8mg/kg×1 was administered. A transient improvement in pyrexia occurredwith administration of corticosteroids on day 5 in CHOP-100, withcomplete resolution of fevers occurring after administration ofcytokine-directed therapy consisting of etanercept and tocilizumab onday 8. FIG. 4B shows serum cytokines and inflammatory markers measuredat frequent time points after CTL019 infusion. Cytokine values are shownusing a semi-logarithmic plot with fold-change from baseline. Baseline(Day 0 pre-infusion) values (pg/ml serum) for each analyte were(CHOP-100, CHOP-101): IL1-β: (0.9, 0.2); IL-6: (4.3, 1.9); TNF-α: (1.5,0.4); IL2Rα: (418.8, 205.7); IL-2: (0.7, 0.4); IL-10 (9.9, 2.3); IL1Rα:(43.9, 27.9). Both patients developed pronounced elevations in a numberof cytokines and cytokine receptors, including soluble interleukin 1Aand 2 receptor (IL-1RA and IL-2R), interleukins 2, 6 and 10 (IL-2, IL-6and IL-10), tumor necrosis factor-α (TNF-α), and interferon-γ (INF-γ).FIG. 4C shows changes in circulating absolute neutrophil count (ANC),absolute lymphocyte count (ALC) and white blood cell (WBC) count. Ofnote, the increase in the ALC was primarily composed of activated CT019T lymphocytes.

FIG. 5, comprising FIGS. 5A through 5D, is a series of imaged depictingexpansion and visualization of CTL019 cells in peripheral blood, bonemarrow and CSF. FIG. 5A shows flow cytometric analysis of peripheralblood stained with antibodies to detect CD3 and the anti-CD19 CAR.Depicted are the percent of CD3 cells expressing the CAR in CHOP-100 andCHOP-101. FIG. 5B shows the presence of CTL019 T cells in peripheralblood, bone marrow, and CSF by quantitative real-time PCR. Genomic DNAwas isolated from whole blood, bone marrow aspirates and CSF collectedat serial time points before and after CTL019 infusion. FIG. 5C showsflow cytometric detection of CTL019 cells in CSF collected from CHOP-100and CHOP-101. FIG. 5D shows images of activated large granularlymphocytes in Wright-stained smears of the peripheral blood andcytospins of the CSF.

FIG. 6 is an image showing CD19 expression at baseline and at relapse inCHOP-101. Bone marrow samples from CHOP-101 were obtained prior toCTL019 infusion and at time of relapse 2 months later. Mononuclear cellsisolated from marrow samples were stained for CD45, CD34 and CD19 andanalyzed on an Accuri C6 flow cytometer. After gating on live cells, theblast gate (CD45+SSC low) was subgated on CD34+ cells and histogramsgenerated for CD19 expression. Division line represents threshold forthe same gating on isotype controls. Pre-therapy blasts have a range ofdistribution of CD19, with a small population of very dim staining cellsseen as the tail of the left histogram at 102 on the X-axis. The relapsesample does not have any CD19 positive blasts. Analysis of CD19expression on the pre-treatment blast population revealed a smallpopulation of CD19 dim or negative cells. The mean fluorescenceintensity (MFI) of this small population of cells was 187 (left panel),similar to the MFI of the anti-CD19-stained relapsed blast cells (201,right panel). Pre-therapy marrow sample was hypocellular with 10% blastsand relapse marrowsample was normocellular with 68% blasts, accountingfor differences in events available for acquisition.

FIG. 7 is an image showing induction of remission in bone marrow inCHOP-101 on day+23 after CTL019 infusion. Clinical immunophenotypingreport for CHOP-101 at baseline (Top panel) and at day+23 (Bottompanel). Cells were stained for CD10, CD19, CD20, CD34, CD38 and CD58.Flow cytometry was done after lysis of the red blood cells. The reporton day+23 indicated that the white blood cells consisted of 42.0%lymphocytes, 6.0% monocytes, 50.3% myeloid forms, 0.17% myeloid blastsand no viable lymphoid progenitors. There was no convincingimmunophenotypic evidence of residual precursor B cell lymphoblasticleukemia/lymphoma by flow cytometry. Essentially no viable B cells wereidentified.

FIG. 8 is an image depicting in vivo expansion and persistence of CTL019cells in blood. The number of white blood cells (WBC), CD3+ T cells, andCTL019 cells in blood is shown for CHOP-100 and CHOP-101. Cell numbersare shown on a semi-logarithmic plot.

FIG. 9, comprising FIGS. 9A and 9B, is a series of images demonstratingthat subjects had an elimination of CD19 positive cells in bone marrowand blood within 1 month after CTL019 infusion. FIG. 9A shows persistentB cell aplasia in CHOP-100. The top panel shows a predominant populationof leukemic blast cells in bone marrow aspirated from CHOP-100expressing CD19 and CD20 on day+6. This population is absent at day+23and 6 months. FIG. 9B shows B cell aplasia and emergence of CD19 escapevariant cells in CHOP-101. Flow cytometric analysis of bone marrowaspirates from CHOP-101 stained with anti-CD45, CD34 and CD19. In thebottom row, side scatter and the CD45 dim positive cells were used toidentify leukemic cells that express variable amounts of CD34 and CD19at baseline. Only CD19 negative blasts were detected on day 64.Numerical values in the top panel represent the fraction of the totalleukocytes represented in each quadrant. Numerical values in the lowerpanel represent the percentage from the total leukocytes represented inthe CD45dim/SS low gate.

FIG. 10 is a graph depicting the levels of ferritin present in thepatient following receipt of CAR T cells.

FIG. 11 is a graph depicting the levels of myoglobin present in thepatient following receipt of CAR T cells.

FIG. 12 is a graph depicting the levels of plasminogen activatorinhibitor-1 (PAI-1) present in the patient following receipt of CARTcells.

DETAILED DESCRIPTION

The invention relates to compositions and methods for treating cancerincluding but not limited to hematologic malignancies and solid tumors.The invention also encompasses methods of treating and preventingcertain types of cancer, including primary and metastatic cancer, aswell as cancers that are refractory or resistant to conventionalchemotherapy. The methods comprise administering to a patient in need ofsuch treatment or prevention a therapeutically or prophylacticallyeffective amount of a T cell transduced to express a chimeric antigenreceptor (CAR). CARs are molecules that combine antibody-basedspecificity for a desired antigen (e.g., tumor antigen) with a T cellreceptor-activating intracellular domain to generate a chimeric proteinthat exhibits a specific anti-tumor cellular immune activity.

As part of the overall treatment regimen, the invention encompassesmethods of managing certain cancers (e.g., preventing or prolongingtheir recurrence, or lengthening the time of remission) by evaluatingthe profile of soluble factors in patients post T cell infusion.Preferably, the profile of soluble factors includes evaluation of acytokine profile. When the cytokine profile indicates an increase in aparticular cytokine post T cell infusion compared to pre T cellinfusion, a skilled artisan can elect to administer to the patient inneed of such management an effective amount of a cytokine inhibitorycompound or a pharmaceutically acceptable salt, solvate, hydrate,stereoisomer, clathrate, or pro drug thereof to manage the elevatedlevels of the cytokine post T cell infusion.

The present invention is partly based on the discovery that the identifyof a unique combination of factors whose modulation from baseline orpre-existing levels at baseline can help track T cell activation, targetactivity, and potential harmful side effects following CAR T cellinfusion in order to help manage the treatment of the cancer. Exemplaryfactors include but are not limited to IL-1β, IL-2, IL-4, IL-5, IL-6,IL-8, IL-10, IL-12, IL-13, IL-15, IL-17, IL-1Rα, IL-2R, IFN-α, IFN-γ,MIP-1α, MIP-1β, MCP-1, TNFα, GM-CSF, G-CSF, CXCL9, CXCL10, CXCR factors,VEGF, RANTES, EOTAXIN, EGF, HGF, FGF-β, CD40, CD40L, ferritin, and thelike.

The present invention relates to a strategy of adoptive cell transfer ofT cells transduced to express a chimeric antigen receptor (CAR) incombination with toxicity management, where a profile of soluble factorsfrom a post T cell infusion patient is generated and a therapy directedagainst the elevated soluble factor is carried out in order to treat thecancer. For example, generating a real time soluble factor profileallows for intervention of the elevated soluble factors with theappropriate inhibitor in order to bring the levels down to normallevels.

In one embodiment, the CAR of the invention comprises an extracellulardomain having an antigen recognition domain that targets a desiredantigen, a transmembrane domain, and a cytoplasmic domain. The inventionis not limited to a specific CAR. Rather, any CAR that targets a desiredantigen can be used in the present invention. Compositions and methodsof making CARs have been described in PCT/US11/64191, which isincorporated by reference in its entirety herein.

In some embodiments of any of the methods above, the methods result in ameasurable reduction in tumor size or evidence of disease or diseaseprogression, complete response, partial response, stable disease,increase or elongation of progression free survival, increase orelongation of overall survival, or reduction in toxicity.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention pertains. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice for testing of the present invention, the preferredmaterials and methods are described herein. In describing and claimingthe present invention, the following terminology will be used.

It is also to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

“About” as used herein when referring to a measurable value such as anamount, a temporal duration, and the like, is meant to encompassvariations of ±20% or ±10%, in some instances ±5%, in some instances±1%, and in some instances ±0.1% from the specified value, as suchvariations are appropriate to perform the disclosed methods.

“Activation,” as used herein, refers to the state of a T cell that hasbeen sufficiently stimulated to induce detectable cellularproliferation. Activation can also be associated with induced cytokineproduction, and detectable effector functions. The term “activated Tcells” refers to, among other things, T cells that are undergoing celldivision.

“Activators” or “agonists” of a soluble factor are used herein to referto molecules of agents capable of activating or increasing the levels ofthe soluble factor. Activators are compounds that increase, promote,induce activation, activate, or upregulate the activity or expression ofsoluble factor, e.g., agonists. Assays for detecting activators include,e.g., expressing the soluble factor in vitro, in cells, or cellmembranes, applying putative agonist compounds, and then determining thefunctional effects on activity of the soluble factor, as describedelsewhere herein.

The term “antibody,” as used herein, refers to an immunoglobulinmolecule which specifically binds with an antigen. Antibodies can beintact immunoglobulins derived from natural sources or from recombinantsources and can be immunoreactive portions of intact immunoglobulins.Antibodies are often tetramers of immunoglobulin molecules. Theantibodies in the present invention may exist in a variety of formsincluding, for example, polyclonal antibodies, monoclonal antibodies,Fv, Fab and F(ab)₂, as well as single chain antibodies and humanizedantibodies (Harlow et al., 1999, In: Using Antibodies: A LaboratoryManual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989,In: Antibodies: A Laboratory Manual, Cold Spring Harbor, N.Y.; Houstonet al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al.,1988, Science 242:423-426).

The term “antibody fragment” refers to a portion of an intact antibodyand refers to the antigenic determining variable regions of an intactantibody. Examples of antibody fragments include, but are not limitedto, Fab, Fab′, F(ab′)2, and Fv fragments, linear antibodies, scFvantibodies, and multispecific antibodies formed from antibody fragments.

The term “antigen” or “Ag” as used herein is defined as a molecule thatprovokes an immune response. This immune response may involve eitherantibody production, or the activation of specificimmunologically-competent cells, or both. The skilled artisan willunderstand that any macromolecule, including virtually all proteins orpeptides, can serve as an antigen. Furthermore, antigens can be derivedfrom recombinant or genomic DNA. A skilled artisan will understand thatany DNA, which comprises a nucleotide sequences or a partial nucleotidesequence encoding a protein that elicits an immune response thereforeencodes an “antigen” as that term is used herein. Furthermore, oneskilled in the art will understand that an antigen need not be encodedsolely by a full length nucleotide sequence of a gene. It is readilyapparent that the present invention includes, but is not limited to, theuse of partial nucleotide sequences of more than one gene and that thesenucleotide sequences are arranged in various combinations to elicit thedesired immune response. Moreover, a skilled artisan will understandthat an antigen need not be encoded by a “gene” at all. It is readilyapparent that an antigen can be generated synthesized or can be derivedfrom a biological sample. Such a biological sample can include, but isnot limited to a tissue sample, a tumor sample, a cell or a biologicalfluid.

The term “auto-antigen” means, in accordance with the present invention,any self-antigen which is recognized by the immune system as if it wereforeign. Auto-antigens comprise, but are not limited to, cellularproteins, phosphoproteins, cellular surface proteins, cellular lipids,nucleic acids, glycoproteins, including cell surface receptors.

The term “autoimmune disease” as used herein is defined as a disorderthat results from an autoimmune response. An autoimmune disease is theresult of an inappropriate and excessive response to a self-antigen.Examples of autoimmune diseases include but are not limited to,Addision's disease, alopecia greata, ankylosing spondylitis, autoimmunehepatitis, autoimmune parotitis, Crohn's disease, diabetes (Type I),dystrophic epidermolysis bullosa, epididymitis, glomerulonephritis,Graves' disease, Guillain-Barr syndrome, Hashimoto's disease, hemolyticanemia, systemic lupus erythematosus, multiple sclerosis, myastheniagravis, pemphigus vulgaris, psoriasis, rheumatic fever, rheumatoidarthritis, sarcoidosis, scleroderma, Sjogren's syndrome,spondyloarthropathies, thyroiditis, vasculitis, vitiligo, myxedema,pernicious anemia, ulcerative colitis, among others.

As used herein, the term “autologous” is meant to refer to any materialderived from the same individual to which it is later to bere-introduced into the individual.

“Allogeneic” refers to a graft derived from a different animal of thesame species.

“Xenogeneic” refers to a graft derived from an animal of a differentspecies.

The term “cancer” as used herein is defined as disease characterized bythe rapid and uncontrolled growth of aberrant cells. Cancer cells canspread locally or through the bloodstream and lymphatic system to otherparts of the body. Examples of various cancers include but are notlimited to, breast cancer, prostate cancer, ovarian cancer, cervicalcancer, skin cancer, pancreatic cancer, colorectal cancer, renal cancer,liver cancer, brain cancer, lymphoma, leukemia, lung cancer and thelike.

As used herein, by “combination therapy” is meant that a first agent isadministered in conjunction with another agent. “In conjunction with”refers to administration of one treatment modality in addition toanother treatment modality. As such, “in conjunction with” refers toadministration of one treatment modality before, during, or afterdelivery of the other treatment modality to the individual. Suchcombinations are considered to be part of a single treatment regimen orregime.

As used herein, the term “concurrent administration” means that theadministration of the first therapy and that of a second therapy in acombination therapy overlap with each other.

“Co-stimulatory ligand,” as the term is used herein, includes a moleculeon an antigen presenting cell (e.g., an aAPC, dendritic cell, B cell,and the like) that specifically binds a cognate co-stimulatory moleculeon a T cell, thereby providing a signal which, in addition to theprimary signal provided by, for instance, binding of a TCR/CD3 complexwith an MHC molecule loaded with peptide, mediates a T cell response,including, but not limited to, proliferation, activation,differentiation, and the like. A co-stimulatory ligand can include, butis not limited to, CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL,OX40L, inducible costimulatory ligand (ICOS-L), intercellular adhesionmolecule (ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM,lymphotoxin beta receptor, 3/TR6, ILT3, ILT4, HVEM, an agonist orantibody that binds Toll ligand receptor and a ligand that specificallybinds with B7-H3. A co-stimulatory ligand also encompasses, inter alia,an antibody that specifically binds with a co-stimulatory moleculepresent on a T cell, such as, but not limited to, CD27, CD28, 4-1BB,OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1(LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specificallybinds with CD83.

A “co-stimulatory molecule” refers to the cognate binding partner on a Tcell that specifically binds with a co-stimulatory ligand, therebymediating a co-stimulatory response by the T cell, such as, but notlimited to, proliferation. Co-stimulatory molecules include, but are notlimited to an MHC class I molecule, BTLA and a Toll ligand receptor.

A “co-stimulatory signal,” as used herein, refers to a signal, which incombination with a primary signal, such as TCR/CD3 ligation, leads to Tcell proliferation and/or upregulation or downregulation of keymolecules.

A “disease” is a state of health of an animal wherein the animal cannotmaintain homeostasis, and wherein if the disease is not ameliorated thenthe animal's health continues to deteriorate. In contrast, a “disorder”in an animal is a state of health in which the animal is able tomaintain homeostasis, but in which the animal's state of health is lessfavorable than it would be in the absence of the disorder. Leftuntreated, a disorder does not necessarily cause a further decrease inthe animal's state of health.

An “effective amount” as used herein, means an amount which provides atherapeutic or prophylactic benefit.

As used herein “endogenous” refers to any material from or producedinside an organism, cell, tissue or system.

As used herein, the term “exogenous” refers to any material introducedto an organism, cell, tissue or system that was produced outside theorganism, cell, tissue or system.

The term “expression” as used herein is defined as the transcriptionand/or translation of a particular nucleotide sequence driven by itspromoter.

“Expression vector” refers to a vector comprising a recombinantpolynucleotide comprising expression control sequences operativelylinked to a nucleotide sequence to be expressed. An expression vectorcomprises sufficient cis-acting elements for expression; other elementsfor expression can be supplied by the host cell or in an in vitroexpression system. Expression vectors include all those known in theart, such as cosmids, plasmids (e.g., naked or contained in liposomes)and viruses (e.g., lentiviruses, retroviruses, adenoviruses, andadeno-associated viruses) that incorporate the recombinantpolynucleotide.

“Homologous” refers to the sequence similarity or sequence identitybetween two polypeptides or between two nucleic acid molecules. When aposition in both of the two compared sequences is occupied by the samebase or amino acid monomer subunit, e.g., if a position in each of twoDNA molecules is occupied by adenine, then the molecules are homologousat that position. The percent of homology between two sequences is afunction of the number of matching or homologous positions shared by thetwo sequences divided by the number of positions compared×100. Forexample, if 6 of 10 of the positions in two sequences are matched orhomologous then the two sequences are 60% homologous. By way of example,the DNA sequences ATTGCC and TATGGC share 50% homology. Generally, acomparison is made when two sequences are aligned to give maximumhomology.

The term “immunoglobulin” or “Ig,” as used herein, is defined as a classof proteins, which function as antibodies. Antibodies expressed by Bcells are sometimes referred to as the BCR (B cell receptor) or antigenreceptor. The five members included in this class of proteins are IgA,IgG, IgM, IgD, and IgE. IgA is the primary antibody that is present inbody secretions, such as saliva, tears, breast milk, gastrointestinalsecretions and mucus secretions of the respiratory and genitourinarytracts. IgG is the most common circulating antibody. IgM is the mainimmunoglobulin produced in the primary immune response in most subjects.It is the most efficient immunoglobulin in agglutination, complementfixation, and other antibody responses, and is important in defenseagainst bacteria and viruses. IgD is the immunoglobulin that has noknown antibody function, but may serve as an antigen receptor. IgE isthe immunoglobulin that mediates immediate hypersensitivity by causingrelease of mediators from mast cells and basophils upon exposure toallergen.

As used herein, the term “immune response” includes T cell mediatedand/or B cell mediated immune responses. Exemplary immune responsesinclude T cell responses, e.g., cytokine production and cellularcytotoxicity. In addition, the term immune response includes immuneresponses that are indirectly effected by T cell activation, e.g.,antibody production (humoral responses) and activation of cytokineresponsive cells, e.g., macrophages. Immune cells involved in the immuneresponse include lymphocytes, such as B cells and T cells (CD4+, CD8+,Th1 and Th2 cells); antigen presenting cells (e.g., professional antigenpresenting cells such as dendritic cells, macrophages, B lymphocytes,Langerhans cells, and non-professional antigen presenting cells such askeratinocytes, endothelial cells, astrocytes, fibroblasts,oligodendrocytes); natural killer cells; myeloid cells, such asmacrophages, eosinophils, mast cells, basophils, and granulocytes.

“Inhibitors” or “antagonists” of a soluble factor are used herein torefer to molecules of agents capable of inhibiting, inactivating orreducing the levels of the soluble factor. Inhibitors are compoundsthat, e.g., bind to, partially or totally block activity, decrease,prevent, delay activation, inactivate, desensitize, or down regulate theactivity or expression of soluble factor, e.g., antagonists. Inhibitorsinclude polypeptide inhibitors, such as antibodies, soluble receptorsand the like, as well as nucleic acid inhibitors such as siRNA orantisense RNA, genetically modified versions of the soluble factor,e.g., versions with altered activity, as well as naturally occurring andsynthetic soluble factor antagonists, small chemical molecules and thelike. Assays for detecting inhibitors include, e.g., expressing thesoluble factor in vitro, in cells, or cell membranes, applying putativeantagonist compounds, and then determining the functional effects onactivity of the soluble factor, as described elsewhere herein.

As used herein, an “instructional material” includes a publication, arecording, a diagram, or any other medium of expression which can beused to communicate the usefulness of the compositions and methods ofthe invention. The instructional material of the kit of the inventionmay, for example, be affixed to a container which contains the nucleicacid, peptide, and/or composition of the invention or be shippedtogether with a container which contains the nucleic acid, peptide,and/or composition. Alternatively, the instructional material may beshipped separately from the container with the intention that theinstructional material and the compound be used cooperatively by therecipient.

“Isolated” means altered or removed from the natural state. For example,a nucleic acid or a peptide naturally present in a living animal is not“isolated,” but the same nucleic acid or peptide partially or completelyseparated from the coexisting materials of its natural state is“isolated.” An isolated nucleic acid or protein can exist insubstantially purified form, or can exist in a non-native environmentsuch as, for example, a host cell.

A “lentivirus” as used herein refers to a genus of the Retroviridaefamily. Lentiviruses are unique among the retroviruses in being able toinfect non-dividing cells; they can deliver a significant amount ofgenetic information into the DNA of the host cell, so they are one ofthe most efficient methods of a gene delivery vector. HIV, SIV, and FIVare all examples of lentiviruses. Vectors derived from lentivirusesoffer the means to achieve significant levels of gene transfer in vivo.

The phrase “level of a soluble factor” in a biological sample as usedherein typically refers to the amount of protein, protein fragment orpeptide levels of the soluble factor that is present in a biologicalsample. A “level of a soluble factor” need not be quantified, but cansimply be detected, e.g., a subjective, visual detection by a human,with or without comparison to a level from a control sample or a levelexpected of a control sample.

By the term “modulating,” as used herein, is meant mediating adetectable increase or decrease in the level of a response in a subjectcompared with the level of a response in the subject in the absence of atreatment or compound, and/or compared with the level of a response inan otherwise identical but untreated subject. The term encompassesperturbing and/or affecting a native signal or response therebymediating a beneficial therapeutic response in a subject, preferably, ahuman.

“Parenteral” administration of an immunogenic composition includes,e.g., subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), orintrasternal injection, or infusion techniques.

The terms “patient,” “subject,” “individual,” and the like are usedinterchangeably herein, and refer to any animal, or cells thereofwhether in vitro or in situ, amenable to the methods described herein.In certain non-limiting embodiments, the patient, subject or individualis a human.

The term “simultaneous administration,” as used herein, means that afirst therapy and second therapy in a combination therapy areadministered with a time separation of no more than about 15 minutes,such as no more than about any of 10, 5, or 1 minutes. When the firstand second therapies are administered simultaneously, the first andsecond therapies may be contained in the same composition (e.g., acomposition comprising both a first and second therapy) or in separatecompositions (e.g., a first therapy in one composition and a secondtherapy is contained in another composition).

The term “simultaneous administration,” as used herein, means that afirst therapy and second therapy in a combination therapy areadministered with a time separation of no more than about 15 minutes,such as no more than about any of 10, 5, or 1 minutes. When the firstand second therapies are administered simultaneously, the first andsecond therapies may be contained in the same composition (e.g., acomposition comprising both a first and second therapy) or in separatecompositions (e.g., a first therapy in one composition and a secondtherapy is contained in another composition).

By the term “specifically binds,” as used herein with respect to anantibody, is meant an antibody which recognizes a specific antigen, butdoes not substantially recognize or bind other molecules in a sample.For example, an antibody that specifically binds to an antigen from onespecies may also bind to that antigen from one or more species. But,such cross-species reactivity does not itself alter the classificationof an antibody as specific. In another example, an antibody thatspecifically binds to an antigen may also bind to different allelicforms of the antigen. However, such cross reactivity does not itselfalter the classification of an antibody as specific. In some instances,the terms “specific binding” or “specifically binding,” can be used inreference to the interaction of an antibody, a protein, or a peptidewith a second chemical species, to mean that the interaction isdependent upon the presence of a particular structure (e.g., anantigenic determinant or epitope) on the chemical species; for example,an antibody recognizes and binds to a specific protein structure ratherthan to proteins generally. If an antibody is specific for epitope “A,”the presence of a molecule containing epitope A (or free, unlabeled A),in a reaction containing labeled “A” and the antibody, will reduce theamount of labeled A bound to the antibody.

By the term “stimulation,” is meant a primary response induced bybinding of a stimulatory molecule (e.g., a TCR/CD3 complex) with itscognate ligand thereby mediating a signal transduction event, such as,but not limited to, signal transduction via the TCR/CD3 complex.Stimulation can mediate altered expression of certain molecules, such asdownregulation of TGF-β, and/or reorganization of cytoskeletalstructures, and the like.

A “stimulatory molecule,” as the term is used herein, means a moleculeon a T cell that specifically binds with a cognate stimulatory ligandpresent on an antigen presenting cell.

A “stimulatory ligand,” as used herein, means a ligand that when presenton an antigen presenting cell (e.g., an aAPC, a dendritic cell, aB-cell, and the like) can specifically bind with a cognate bindingpartner (referred to herein as a “stimulatory molecule”) on a T cell,thereby mediating a primary response by the T cell, including, but notlimited to, activation, initiation of an immune response, proliferation,and the like. Stimulatory ligands are well-known in the art andencompass, inter alia, an MHC Class I molecule loaded with a peptide, ananti-CD3 antibody, a superagonist anti-CD28 antibody, and a superagonistanti-CD2 antibody.

The term “subject” is intended to include living organisms in which animmune response can be elicited (e.g., mammals). Examples of subjectsinclude humans, dogs, cats, mice, rats, and transgenic species thereof.

As used herein, a “substantially purified” cell is a cell that isessentially free of other cell types. A substantially purified cell alsorefers to a cell which has been separated from other cell types withwhich it is normally associated in its naturally occurring state. Insome instances, a population of substantially purified cells refers to ahomogenous population of cells. In other instances, this term referssimply to cell that have been separated from the cells with which theyare naturally associated in their natural state. In some embodiments,the cells are cultured in vitro. In other embodiments, the cells are notcultured in vitro.

The term “therapeutic” as used herein means a treatment and/orprophylaxis. A therapeutic effect is obtained by suppression, remission,or eradication of a disease state.

The term “therapeutically effective amount” refers to the amount of thesubject compound that will elicit the biological or medical response ofa tissue, system, or subject that is being sought by the researcher,veterinarian, medical doctor or other clinician. The term“therapeutically effective amount” includes that amount of a compoundthat, when administered, is sufficient to prevent development of, oralleviate to some extent, one or more of the signs or symptoms of thedisorder or disease being treated. The therapeutically effective amountwill vary depending on the compound, the disease and its severity andthe age, weight, etc., of the subject to be treated.

A “transplant,” as used herein, refers to cells, tissue, or an organthat is introduced into an individual. The source of the transplantedmaterial can be cultured cells, cells from another individual, or cellsfrom the same individual (e.g., after the cells are cultured in vitro).Exemplary organ transplants are kidney, liver, heart, lung, andpancreas.

To “treat” a disease as the term is used herein, means to reduce thefrequency or severity of at least one sign or symptom of a disease ordisorder experienced by a subject.

The term “transfected” or “transformed” or “transduced” as used hereinrefers to a process by which exogenous nucleic acid is transferred orintroduced into the host cell. A “transfected” or “transformed” or“transduced” cell is one which has been transfected, transformed ortransduced with exogenous nucleic acid. The cell includes the primarysubject cell and its progeny.

Ranges: throughout this disclosure, various aspects of the invention canbe presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. Thisapplies regardless of the breadth of the range.

DESCRIPTION

The present invention provides compositions and methods for treatingcancer in a patient. In one embodiment, the treatment method comprises afirst-line of therapy comprising administering the CAR of the inventioninto the patient to induce an anti-tumor immune response and monitoringthe levels of soluble factors in the patient post T cell infusion todetermine the type of second-line of therapy appropriate to treat thepatient as a consequence of the first-line of therapy.

In one embodiment, the second-line of therapy comprises evaluating theprofile of soluble factors in a patient following receipt of an infusionof the appropriate CAR T (referred elsewhere herein as “post T cellinfusion”) where when the soluble factor profile indicates an increasein a particular soluble factor post T cell infusion compared to pre Tcell infusion, a skilled artisan can elect to administer to the patientin need of an effective amount of a soluble factor inhibitory compoundin order to manage the elevated levels of the soluble factor post T cellinfusion. Accordingly, the second-line of therapy in one embodimentincludes administering a type of soluble factor inhibitory therapy tomanage the elevated levels of certain soluble factor s resulting fromthe first-line of therapy of using CART cells.

In yet another embodiment, the second-line of therapy relating toadministering a soluble factor inhibitory compound to the patient can becombined with other conventionally therapies used to treat, prevent ormanage diseases or disorders associated with, or characterized by,undesired angiogenesis. Examples of such conventional therapies include,but are not limited to, surgery, chemotherapy, radiation therapy,hormonal therapy, biological therapy and immunotherapy.

In one embodiment, the CAR of the invention can be engineered tocomprise an extracellular domain having an antigen binding domain thattargets tumor antigen fused to an intracellular signaling domain of theT cell antigen receptor complex zeta chain (e.g., CD3 zeta). Anexemplary tumor antigen B cell antigen is CD19 because this antigen isexpressed on malignant B cells. However, the invention is not limited totargeting CD19. Rather, the invention includes any tumor antigen bindingmoiety. The antigen binding moiety is preferably fused with anintracellular domain from one or more of a costimulatory molecule and azeta chain. Preferably, the antigen binding moiety is fused with one ormore intracellular domains selected from the group of a CD137 (4-1BB)signaling domain, a CD28 signaling domain, a CD3zeta signal domain, andany combination thereof.

In one embodiment, the CAR of the invention comprises a CD137 (4-1BB)signaling domain. This is because the present invention is partly basedon the discovery that CAR-mediated T-cell responses can be furtherenhanced with the addition of costimulatory domains. For example,inclusion of the CD137 (4-1BB) signaling domain significantly increasedCAR mediated activity and in vivo persistence of CAR T cells compared toan otherwise identical CAR T cell not engineered to express CD137(4-1BB). However, the invention is not limited to a specific CAR.Rather, any CAR that targets a tumor antigen can be used in the presentinvention. Compositions and methods of making and using CARs have beendescribed in PCT/US11/64191, which is incorporated by reference in itsentirety herein.

Methods

The treatment regimen of the invention result in a measurable reductionin tumor size or evidence of disease or disease progression, completeresponse, partial response, stable disease, increase or elongation ofprogression free survival, increase or elongation of overall survival,or reduction in toxicity.

As part of the overall treatment regimen, the invention encompasses afirst-line and a second-line therapy, wherein the first-line therapycomprises administering a CAR T cell of the invention to the patient inneed thereof. The treatment regimen of the invention allows for themanagement of the cancer and treatment thereof by evaluating the solublefactor profile in patients post T cell infusion. An appropriatesecond-line therapy comprises administering an appropriate solublefactor inhibitor to the patient in order to reduce the elevated levelsof the soluble factor resulting from the first-line therapy. In someinstances, the appropriate second-line therapy comprises administeringan appropriate soluble factor activator to the patient in order toincrease the suppressed levels of the soluble factor resulting from thefirst-line therapy.

In one embodiment, an appropriate second-line therapy comprisesadministering an appropriate cytokine inhibitor to the patient in orderto reduce the elevated levels of the cytokine resulting from thefirst-line therapy. In some instances, the appropriate second-linetherapy comprises administering an appropriate cytokine activator to thepatient in order to increase the suppressed levels of the cytokineresulting from the first-line therapy.

In one embodiment, differential levels are over expression (highexpression) or under expression (low expression) as compared to theexpression level of a normal or control cell, a given patientpopulation, or with an internal control. In some embodiments, levels arecompared between the patient and a normal individual, between thepatient post T cell infusion and pre T cell infusion, or between thepatient post T cell infusion at a first time point and a second timepoint.

In one embodiment, the invention includes evaluating differential levelsof one or more cytokines to generate a cytokine profile in a patientpost T cell infusion in order to determine the type of cytokine therapyto be applied to the patient for the purpose of regulating the cytokinelevel back to normal levels. The invention may therefore be applied toidentify cytokine levels elevated as a result of the presence of theCART cells of the invention in the patient, which allows the specializedtreatment of the patient with cytokine inhibitors to decrease theelevated levels of the cytokine. In another embodiment, invention may beapplied to identify cytokine levels decreased as a result of thepresence of the CART cells of the invention in the patient, which allowsthe specialized treatment of the patient with cytokine activators toincrease the diminished levels of the cytokine.

In one embodiment, cytokines levels that are elevated as a result ofreceiving a CART cell infusion include but are not limited to IL-1β,IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, IL-13, IL-15, IL-17, IL-1Rα,IL-2R, IFN-α, IFN-γ, MIP-1α, MIP-1β, MCP-1, TNFα, GM-CSF, G-CSF, CXCL9,CXCL10, CXCR factors, VEGF, RANTES, EOTAXIN, EGF, HGF, FGF-β, CD40,CD40L, ferritin, and the like. However, the invention should not belimited to these listed cytokines. Rather, the invention includes anycytokine identified to be elevated in a patient as a result of receivinga CAR T cell infusion.

In one embodiment, cytokines levels that are decreased as a result ofreceiving a CART cell infusion include but are not limited to IL-1β,IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, IL-13, IL-15, IL-17, IL-1Rα,IL-2R, IFN-α, IFN-γ, MIP-1α, MIP-1β, MCP-1, TNFα, GM-CSF, G-CSF, CXCL9,CXCL10, CXCR factors, VEGF, RANTES, EOTAXIN, EGF, HGF, FGF-β, CD40,CD40L, ferritin, and the like. However, the invention should not belimited to these listed cytokines. Rather, the invention includes anycytokine identified to be decreased in a patient as a result ofreceiving a CAR T cell infusion.

Detecting a Cytokine and Treatment Thereof

Although this section describes detection of a cytokine and treatmentthereof as part of the second-line therapy, the invention encompassesdetection of any soluble factor and treatment thereof as part of thesecond-line therapy. Therefore, the description in the context of a“cytokine” can equally be applied to a “soluble factor.”

In one embodiment, as part of the second-line therapy, the inventionincludes methods of detecting levels of a cytokine in a patient that hasreceived infusion of a CART cell of the invention. In some embodiments,the presence or level of a cytokine can be used to select a candidatetreatment. In some other embodiments, the presence or levels of thecytokine can be used to determine the success during the course of orafter treatment of the first-line, second-line, or both the first andsecond-line of therapy.

Biological samples in which the cytokine can be detected include, forexample, serum. In some embodiments, biological samples include a tissuebiopsy which may or may not have a liquid component.

Immunoassays can be used to qualitatively or quantitatively analyze thecytokine levels in a biological sample. A general overview of theapplicable technology can be found in a number of readily availablemanuals, e.g., Harlow & Lane, Cold Spring Harbor Laboratory Press, UsingAntibodies: A Laboratory Manual (1999).

In addition to using immunoassays to detect the levels of cytokines in abiological sample from a patient, assessment of cytokine expression andlevels can be made based on the level of gene expression of theparticular cytokines. RNA hybridization techniques for determining thepresence and/or level of mRNA expression are well known to those ofskill in the art and can be used to assess the presence or level of geneexpression of the cytokine of interest.

In some embodiments, the methods of the present invention utilizeselective binding partners of the cytokine to identify the presence ordetermine the levels of the cytokine in a biological sample. Theselective binding partner to be used with the methods and kits of thepresent invention can be, for instance, an antibody. In some aspects,monoclonal antibodies to the particular cytokine can be used. In someother aspects, polyclonal antibodies to the particular cytokine can beemployed to practice the methods and in the kits of the presentinvention.

Commercial antibodies to the cytokine are available and can be used withthe methods and kits of the present invention. It is well known to thoseof skill in the art that the type, source and other aspects of anantibody to be used is a consideration to be made in light of the assayin which the antibody is used. In some instances, antibodies that willrecognize its antigen target on a Western blot might not applicable toan ELISA or ELISpot assay and vice versa.

In some embodiments, the antibodies to be used for the assays of thepresent invention can be produced using techniques for producingmonoclonal or polyclonal antibodies that are well known in the art (see,e.g., Coligan, Current Protocols in Immunology (1991); Harlow & Lane,supra; Goding, Monoclonal Antibodies: Principles and Practice (2d ed.1986); and Kohler & Milstein, Nature 256:495-497 (1975). Such techniquesinclude antibody preparation by selection of antibodies from librariesof recombinant antibodies in phage or similar vectors, as well aspreparation of polyclonal and monoclonal antibodies by immunizingrabbits or mice (see, e.g., Huse et al., Science 246:1275-1281 (1989);Ward et al., Nature 341:544-546 (1989)). Such antibodies can be used fortherapeutic and diagnostic applications, e.g., in the treatment and/ordetection of any of the specific cytokine-associated diseases orconditions described herein.

Detection methods employing immunoassays are particularly suitable forpractice at the point of patient care. Such methods allow for immediatediagnosis and/or prognostic evaluation of the patient. Point of carediagnostic systems are described, e.g., in U.S. Pat. No. 6,267,722 whichis incorporated herein by reference. Other immunoassay formats are alsoavailable such that an evaluation of the biological sample can beperformed without having to send the sample to a laboratory forevaluation. Typically these assays are formatted as solid assays where areagent, e.g., an antibody is used to detect the cytokine. Exemplarytest devices suitable for use with immunoassays such as assays of thepresent invention are described, for example, in U.S. Pat. Nos.7,189,522; 6,818,455 and 6,656,745.

In some aspects, the present invention provides methods for detection ofpolynucleotide sequences which code for the cytokine in a biologicalsample. As noted above, a “biological sample” refers to a cell orpopulation of cells or a quantity of tissue or fluid from a patient.Most often, the sample has been removed from a patient, but the term“biological sample” can also refer to cells or tissue analyzed in vivo,i.e., without removal from the patient. Typically, a “biological sample”will contain cells from the patient, but the term can also refer tononcellular biological material.

In one embodiment, amplification-based assays are used to measure thelevel of a desired cytokine. In such an assay, nucleic acid sequences ofthe desired cytokine act as a template in an amplification reaction(e.g., Polymerase Chain Reaction, or PCR). In a quantitativeamplification, the amount of amplification product will be proportionalto the amount of template in the original sample. Comparison toappropriate controls provides a measure of the copy number of thecytokine associated gene. Methods of quantitative amplification are wellknown to those of skill in the art. Detailed protocols for quantitativePCR are provided, e.g., in Innis et al. (1990) PCR Protocols, A Guide toMethods and Applications, Academic Press, Inc. N.Y.). RT-PCR methods arewell known to those of skill (see, e.g., Ausubel et al., supra). In someembodiments, quantitative RT-PCR, e.g., a TaqMan™ assay, is used,thereby allowing the comparison of the level of mRNA in a sample with acontrol sample or value. The known nucleic acid sequences for a desiredcytokine are sufficient to enable one of skill to routinely selectprimers to amplify any portion of the gene. Suitable primers foramplification of specific sequences can be designed using principleswell known in the art (see, e.g., Dieffenfach & Dveksler, PCR Primer: ALaboratory Manual (1995)).

In some embodiments, hybridization based assays can be used to detectthe amount of a desired cytokine in the cells of a biological sample.Such assays include dot blot analysis of RNA as well as other assays,e.g., fluorescent in situ hybridization, which is performed on samplesthat comprise cells. Other hybridization assays are readily available inthe art.

In numerous embodiments of the present invention, the level and/orpresence of a cytokine polynucleotide or polypeptide will be detected ina biological sample, thereby detecting the differential expression ofthe cytokine to generate a cytokine profile from a biological samplederived from a patient infused with a CAR T cell of the inventioncompared to the control biological sample.

The amount of a cytokine polynucleotide or polypeptide detected in thebiological sample indicates the presence of a cytokine to generate acytokine profile for the purpose of classifying the patient for theappropriate cytokine treatment. For example, when the cytokine profileindicates an increase in a particular cytokine post T cell infusioncompared to control (e.g., pre T cell infusion), a skilled artisan canelect to administer to the patient in need of such management aneffective amount of a cytokine inhibitory compound. Alternatively, whenthe cytokine profile indicates a decrease in a particular cytokine postT cell infusion compared to control (e.g., pre T cell infusion), askilled artisan can elect to administer to the patient in need of suchmanagement an effective amount of a cytokine activator compound.

In some embodiments, the difference in cytokine levels between the postT cell infusion sample and the control sample and be at least about 0.5,1.0, 1.5, 2, 5, 10, 100, 200, 500, 1000 fold.

The present methods can also be used to assess the efficacy of a courseof treatment. For example, in a post T cell infusion patient containingan elevated amount of a cytokine IL-6, the efficacy of an anti-IL-6treatment can be assessed by monitoring, over time, IL-6. For example, areduction in IL-6 polynucleotide or polypeptide levels in a biologicalsample taken from a patient following a treatment, compared to a levelin a sample taken from the mammal before, or earlier in, the treatment,indicates efficacious treatment.

In one embodiment, a treatment regimen can be based on neutralizing theelevated cytokine. For example, antagonists of a cytokine can beselected for treatment. Antibodies are an example of a suitableantagonist and include mouse antibodies, chimeric antibodies, humanizedantibodies, and human antibodies or fragments thereof. Chimericantibodies are antibodies whose light and heavy chain genes have beenconstructed, typically by genetic engineering, from immunoglobulin genesegments belonging to different species (see, e.g., Boyce et al., Annalsof Oncology 14:520-535 (2003)). For example, the variable (V) segmentsof the genes from a mouse monoclonal antibody may be joined to humanconstant (C) segments. A typical chimeric antibody is thus a hybridprotein consisting of the V or antigen-binding domain from a mouseantibody and the C or effector regions from a human antibody.

Humanized antibodies have variable region framework residuessubstantially from a human antibody (termed an acceptor antibody) andcomplementarity determining regions substantially from a mouse-antibody,(referred to as the donor immunoglobulin). See Queen et al., Proc. NatL.Acad. Sci. USA 86:10029-10033 (1989) and WO 90/07861, U.S. Pat. No.5,693,762, U.S. Pat. No. 5,693,761, U.S. Pat. No. 5,585,089, U.S. Pat.No. 5,530,101 and Winter, U.S. Pat. No. 5,225,539. The constantregion(s), if present, are also substantially or entirely from a humanimmunoglobulin. Antibodies can be obtained by conventional hybridomaapproaches, phage display (see, e.g., Dower et al., WO 91/17271 andMcCafferty et al., WO 92/01047), use of transgenic mice with humanimmune systems (Lonberg et al., WO93/12227 (1993)), among other sources.Nucleic acids encoding immunoglobulin chains can be obtained fromhybridomas or cell lines producing antibodies, or based onimmunoglobulin nucleic acid or amino acid sequences in the publishedliterature.

Other antagonists of a desired cytokine can also be used for treatmentpurposes. For example, a class of antagonists that can be used for thepurposes of the present invention, are the soluble forms of thereceptors for the cytokine. By way of merely illustrative purposes, anIL-6 antagonist is an anti-IL-6 antibody that specifically binds toIL-6. A specific antibody has the ability to inhibit or antagonize theaction of IL-6 systemically. In some embodiments, the antibody bindsIL-6 and prevents it from interacting with or activating its receptors(e.g. IL-6Ra or IL-6Rβ). In some embodiments, the activity of IL-6 canbe antagonized by using an antagonist to the interleukin-6 receptors(IL-6R). U.S. Application number 2006251653 describes methods fortreating interleukin-6 related disease and discloses a number ofinterleukin-6 antagonists including, for example, humanized anti-IL-6Rantibodies and chimeric anti-IL-6R antibodies. In some embodiments, anIL-6 or IL-6R derivative can be used to block and antagonize theinteraction between IL-6/IL-6R.

The invention is not limited to the cytokines and their correspondingactivators and inhibitors described herein. Rather, the inventionincludes the used of any cytokine activator and/or inhibitor that isused in the art to modulate the cytokine. This is because the inventionis based on managing cancer treatment in a patient receiving infusion ofCAR T cells of the invention wherein the infused CAR T cells result inincrease and decrease levels of various cytokines. One skilled in theart based on the disclosure presented herein that differentialexpression levels of a cytokine in a post T cell infusion samplecompared to a control sample can be targeted for treatment for have thecytokine level be increased or decreased to normal levels.

Therapeutic Application

The present invention encompasses a cell (e.g., T cell) transduced witha lentiviral vector (LV). For example, the LV encodes a CAR thatcombines an antigen recognition domain of a specific antibody with anintracellular domain of CD3-zeta, CD28, 4-1BB, or any combinationsthereof. Therefore, in some instances, the transduced T cell can elicita CAR-mediated T-cell response.

The invention provides the use of a CAR to redirect the specificity of aprimary T cell to a tumor antigen. Thus, the present invention alsoprovides a method for stimulating a T cell-mediated immune response to atarget cell population or tissue in a mammal comprising the step ofadministering to the mammal a T cell that expresses a CAR, wherein theCAR comprises a binding moiety that specifically interacts with apredetermined target, a zeta chain portion comprising for example theintracellular domain of human CD3zeta, and a costimulatory signalingregion.

In one embodiment, the present invention includes a type of cellulartherapy where T cells are genetically modified to express a CAR and theCAR T cell is infused to a recipient in need thereof. The infused cellis able to kill tumor cells in the recipient. Unlike antibody therapies,CAR T cells are able to replicate in vivo resulting in long-termpersistence that can lead to sustained tumor control.

In one embodiment, the CART cells of the invention can undergo robust invivo T cell expansion and can persist for an extended amount of time. Inanother embodiment, the CAR T cells of the invention evolve intospecific memory T cells that can be reactivated to inhibit anyadditional tumor formation or growth. For example, it was unexpectedthat the CART19 cells of the invention can undergo robust in vivo T cellexpansion and persist at high levels for an extended amount of time inblood and bone marrow and form specific memory T cells. Without wishingto be bound by any particular theory, CAR T cells may differentiate invivo into a central memory-like state upon encounter and subsequentelimination of target cells expressing the surrogate antigen.

Without wishing to be bound by any particular theory, the anti-tumorimmunity response elicited by the CAR-modified T cells may be an activeor a passive immune response. In addition, the CAR mediated immuneresponse may be part of an adoptive immunotherapy approach in whichCAR-modified T cells induce an immune response specific to the antigenbinding moiety in the CAR. For example, a CART19 cells elicits an immuneresponse specific against cells expressing CD19.

While the data disclosed herein specifically disclose lentiviral vectorcomprising anti-CD19 scFv derived from FMC63 murine monoclonal antibody,human CD8a hinge and transmembrane domain, and human 4-1BB and CD3zetasignaling domains, the invention should be construed to include anynumber of variations for each of the components of the construct asdescribed elsewhere herein. That is, the invention includes the use ofany antigen binding moiety in the CAR to generate a CAR-mediated T-cellresponse specific to the antigen binding moiety. For example, theantigen binding moiety in the CAR of the invention can target a tumorantigen for the purposes of treat cancer.

Cancers that may be treated include tumors that are not vascularized, ornot yet substantially vascularized, as well as vascularized tumors. Thecancers may comprise non-solid tumors (such as hematological tumors, forexample, leukemias and lymphomas) or may comprise solid tumors. Types ofcancers to be treated with the CARs of the invention include, but arenot limited to, carcinoma, blastoma, and sarcoma, and certain leukemiaor lymphoid malignancies, benign and malignant tumors, and malignanciese.g., sarcomas, carcinomas, and melanomas. Adult tumors/cancers andpediatric tumors/cancers are also included.

Hematologic cancers are cancers of the blood or bone marrow. Examples ofhematological (or hematogenous) cancers include leukemias, includingacute leukemias (such as acute lymphocytic leukemia, acute myelocyticleukemia, acute myelogenous leukemia and myeloblastic, promyelocytic,myelomonocytic, monocytic and erythroleukemia), chronic leukemias (suchas chronic myelocytic (granulocytic) leukemia, chronic myelogenousleukemia, and chronic lymphocytic leukemia), polycythemia vera,lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and highgrade forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavychain disease, myelodysplastic syndrome, hairy cell leukemia andmyelodysplasia.

Solid tumors are abnormal masses of tissue that usually do not containcysts or liquid areas. Solid tumors can be benign or malignant.Different types of solid tumors are named for the type of cells thatform them (such as sarcomas, carcinomas, and lymphomas). Examples ofsolid tumors, such as sarcomas and carcinomas, include fibrosarcoma,myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, and othersarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,rhabdomyosarcoma, colon carcinoma, lymphoid malignancy, pancreaticcancer, breast cancer, lung cancers, ovarian cancer, prostate cancer,hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma,adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma,papillary thyroid carcinoma, pheochromocytomas sebaceous glandcarcinoma, papillary carcinoma, papillary adenocarcinomas, medullarycarcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bileduct carcinoma, choriocarcinoma, Wilms' tumor, cervical cancer,testicular tumor, seminoma, bladder carcinoma, melanoma, and CNS tumors(such as a glioma (such as brainstem glioma and mixed gliomas),glioblastoma (also known as glioblastoma multiforme) astrocytoma, CNSlymphoma, germinoma, medulloblastoma, Schwannoma craniopharyogioma,ependymoma, pinealoma, hemangioblastoma, acoustic neuroma,oligodendroglioma, menangioma, neuroblastoma, retinoblastoma and brainmetastases).

In one embodiment, the antigen bind moiety portion of the CAR of theinvention is designed to treat a particular cancer. For example, the CARdesigned to target CD19 can be used to treat cancers and disordersincluding but are not limited to pre-B ALL (pediatric indication), adultALL, mantle cell lymphoma, diffuse large B-cell lymphoma, salvage postallogenic bone marrow transplantation, and the like.

In another embodiment, the CAR can be designed to target CD22 to treatdiffuse large B-cell lymphoma.

In one embodiment, cancers and disorders include but are not limited topre-B ALL (pediatric indication), adult ALL, mantle cell lymphoma,diffuse large B-cell lymphoma, salvage post allogenic bone marrowtransplantation, and the like can be treated using a combination of CARsthat target CD19, CD20, CD22, and ROR1.

In one embodiment, the CAR can be designed to target mesothelin to treatmesothelioma, pancreatic cancer, ovarian cancer, and the like.

In one embodiment, the CAR can be designed to target CD33/IL3Ra to treatacute myelogenous leukemia and the like.

In one embodiment, the CAR can be designed to target c-Met to treattriple negative breast cancer, non-small cell lung cancer, and the like.

In one embodiment, the CAR can be designed to target PSMA to treatprostate cancer and the like.

In one embodiment, the CAR can be designed to target Glycolipid F77 totreat prostate cancer and the like.

In one embodiment, the CAR can be designed to target EGFRvIII to treatgliobastoma and the like.

In one embodiment, the CAR can be designed to target GD-2 to treatneuroblastoma, melanoma, and the like.

In one embodiment, the CAR can be designed to target NY-ESO-1 TCR totreat myeloma, sarcoma, melanoma, and the like.

In one embodiment, the CAR can be designed to target MAGE A3 TCR totreat myeloma, sarcoma, melanoma, and the like.

However, the invention should not be construed to be limited to solelyto the antigen targets and diseases disclosed herein. Rather, theinvention should be construed to include any antigenic target that isassociated with a disease where a CAR can be used to treat the disease.

The CAR-modified T cells of the invention may also serve as a type ofvaccine for ex vivo immunization and/or in vivo therapy in a mammal.Preferably, the mammal is a human.

With respect to ex vivo immunization, at least one of the followingoccurs in vitro prior to administering the cell into a mammal: i)expansion of the cells, ii) introducing a nucleic acid encoding a CAR tothe cells, and/or iii) cryopreservation of the cells.

Ex vivo procedures are well known in the art and are discussed morefully below. Briefly, cells are isolated from a mammal (preferably ahuman) and genetically modified (i.e., transduced or transfected invitro) with a vector expressing a CAR disclosed herein. The CAR-modifiedcell can be administered to a mammalian recipient to provide atherapeutic benefit. The mammalian recipient may be a human and theCAR-modified cell can be autologous with respect to the recipient.Alternatively, the cells can be allogeneic, syngeneic or xenogeneic withrespect to the recipient.

The procedure for ex vivo expansion of hematopoietic stem and progenitorcells is described in U.S. Pat. No. 5,199,942, incorporated herein byreference, can be applied to the cells of the present invention. Othersuitable methods are known in the art, therefore the present inventionis not limited to any particular method of ex vivo expansion of thecells. Briefly, ex vivo culture and expansion of T cells comprises: (1)collecting CD34+ hematopoietic stem and progenitor cells from a mammalfrom peripheral blood harvest or bone marrow explants; and (2) expandingsuch cells ex vivo. In addition to the cellular growth factors describedin U.S. Pat. No. 5,199,942, other factors such as flt3-L, IL-1, IL-3 andc-kit ligand, can be used for culturing and expansion of the cells.

In addition to using a cell-based vaccine in terms of ex vivoimmunization, the present invention also provides compositions andmethods for in vivo immunization to elicit an immune response directedagainst an antigen in a patient.

Generally, the cells activated and expanded as described herein may beutilized in the treatment and prevention of diseases that arise inindividuals who are immunocompromised. In particular, the CAR-modified Tcells of the invention are used in the treatment of CCL. In certainembodiments, the cells of the invention are used in the treatment ofpatients at risk for developing CCL. Thus, the present inventionprovides methods for the treatment or prevention of CCL comprisingadministering to a subject in need thereof, a therapeutically effectiveamount of the CAR-modified T cells of the invention.

The CAR-modified T cells of the present invention may be administeredeither alone, or as a pharmaceutical composition in combination withdiluents and/or with other components such as IL-2 or other cytokines orcell populations. Briefly, pharmaceutical compositions of the presentinvention may comprise a target cell population as described herein, incombination with one or more pharmaceutically or physiologicallyacceptable carriers, diluents or excipients. Such compositions maycomprise buffers such as neutral buffered saline, phosphate bufferedsaline and the like; carbohydrates such as glucose, mannose, sucrose ordextrans, mannitol; proteins; polypeptides or amino acids such asglycine; antioxidants; chelating agents such as EDTA or glutathione;adjuvants (e.g., aluminum hydroxide); and preservatives. Compositions ofthe present invention are preferably formulated for intravenousadministration.

Pharmaceutical compositions of the present invention may be administeredin a manner appropriate to the disease to be treated (or prevented). Thequantity and frequency of administration will be determined by suchfactors as the condition of the patient, and the type and severity ofthe patient's disease, although appropriate dosages may be determined byclinical trials.

When “an immunologically effective amount”, “an anti-tumor effectiveamount”, “an tumor-inhibiting effective amount”, or “therapeutic amount”is indicated, the precise amount of the compositions of the presentinvention to be administered can be determined by a physician withconsideration of individual differences in age, weight, tumor size,extent of infection or metastasis, and condition of the patient(subject). It can generally be stated that a pharmaceutical compositioncomprising the T cells described herein may be administered at a dosageof 10⁴ to 10⁹ cells/kg body weight, preferably 10⁵ to 10⁶ cells/kg bodyweight, including all integer values within those ranges. T cellcompositions may also be administered multiple times at these dosages.The cells can be administered by using infusion techniques that arecommonly known in immunotherapy (see, e.g., Rosenberg et al., New Eng.J. of Med. 319:1676, 1988). The optimal dosage and treatment regime fora particular patient can readily be determined by one skilled in the artof medicine by monitoring the patient for signs of disease and adjustingthe treatment accordingly.

In certain embodiments, it may be desired to administer activated Tcells to a subject and then subsequently redraw blood (or have anapheresis performed), activate T cells therefrom according to thepresent invention, and reinfuse the patient with these activated andexpanded T cells. This process can be carried out multiple times everyfew weeks. In certain embodiments, T cells can be activated from blooddraws of from 10 cc to 400 cc. In certain embodiments, T cells areactivated from blood draws of 20 cc, 30 cc, 40 cc, 50 cc, 60 cc, 70 cc,80 cc, 90 cc, or 100 cc. Not to be bound by theory, using this multipleblood draw/multiple reinfusion protocol may serve to select out certainpopulations of T cells.

The administration of the subject compositions may be carried out in anyconvenient manner, including by aerosol inhalation, injection,ingestion, transfusion, implantation or transplantation. Thecompositions described herein may be administered to a patientsubcutaneously, intradermally, intratumorally, intranodally,intramedullary, intramuscularly, by intravenous (i.v.) injection, orintraperitoneally. In one embodiment, the T cell compositions of thepresent invention are administered to a patient by intradermal orsubcutaneous injection. In another embodiment, the T cell compositionsof the present invention are preferably administered by i.v. injection.The compositions of T cells may be injected directly into a tumor, lymphnode, or site of infection.

In certain embodiments of the present invention, cells activated andexpanded using the methods described herein, or other methods known inthe art where T cells are expanded to therapeutic levels, areadministered to a patient in conjunction with (e.g., before,simultaneously or following) any number of relevant treatmentmodalities, including but not limited to treatment with agents such asantiviral therapy, cidofovir and interleukin-2, Cytarabine (also knownas ARA-C) or natalizumab treatment for MS patients or efalizumabtreatment for psoriasis patients or other treatments for PML patients.In further embodiments, the T cells of the invention may be used incombination with chemotherapy, radiation, immunosuppressive agents, suchas cyclosporin, azathioprine, methotrexate, mycophenolate, and FK506,antibodies, or other immunoablative agents such as CAM PATH, anti-CD3antibodies or other antibody therapies, cytoxin, fludaribine,cyclosporin, FK506, rapamycin, mycophenolic acid, steroids, FR901228,cytokines, and irradiation. These drugs inhibit either the calciumdependent phosphatase calcineurin (cyclosporine and FK506) or inhibitthe p70S6 kinase that is important for growth factor induced signaling(rapamycin) (Liu et al., Cell 66:807-815, 1991; Henderson et al., Immun.73:316-321, 1991; Bierer et al., Curr. Opin. Immun. 5:763-773, 1993). Ina further embodiment, the cell compositions of the present invention areadministered to a patient in conjunction with (e.g., before,simultaneously or following) bone marrow transplantation, T cellablative therapy using either chemotherapy agents such as, fludarabine,external-beam radiation therapy (XRT), cyclophosphamide, or antibodiessuch as OKT3 or CAMPATH. In another embodiment, the cell compositions ofthe present invention are administered following B-cell ablative therapysuch as agents that react with CD20, e.g., Rituxan. For example, in oneembodiment, subjects may undergo standard treatment with high dosechemotherapy followed by peripheral blood stem cell transplantation. Incertain embodiments, following the transplant, subjects receive aninfusion of the expanded immune cells of the present invention. In anadditional embodiment, expanded cells are administered before orfollowing surgery.

The dosage of the above treatments to be administered to a patient willvary with the precise nature of the condition being treated and therecipient of the treatment. The scaling of dosages for humanadministration can be performed according to art-accepted practices. Thedose for CAMPATH, for example, will generally be in the range 1 to about100 mg for an adult patient, usually administered daily for a periodbetween 1 and 30 days. The preferred daily dose is 1 to 10 mg per dayalthough in some instances larger doses of up to 40 mg per day may beused (described in U.S. Pat. No. 6,120,766).

Treatment of Cytokine Release Syndrome (CRS)

The invention is based partly on the discovery that in vivoproliferation of CART19 cells and the potent anti-tumor activityassociated therewith is also associated with with CRS, leading tohemophagocytic lymphohistiocytosis (HLH), also termed MacrophageActivation Syndrome (MAS). Without wishing to be bound by any particulartheory, it is believed that that MAS/HLH is a unique biomarker that isassociated with and may be required for CART19 potent anti-tumoractivity.

Accordingly, the invention provides a first-line of therapy comprisingadministering the CAR of the invention into the patient and asecond-line of therapy comprising administering a type of therapy tomanage the elevated levels of certain soluble factors resulting from thefirst-line of therapy of using CAR T cells.

In one embodiment, the second-line of therapy comprises compositions andmethods for the treatment of CRS. Symptoms of CRS include high fevers,nausea, transient hypotension, hypoxia, and the like. The presentinvention is based on the observation that CART19 cells induced elevatedlevels of soluble factors in the patient including but is not limited toIFN-γ, TNFα, IL-2 and IL-6. Therefore, the second-line of therapycomprises compounds and methods for neutralizing the effects against theelevated cytokines resulting from the administration of the CART19cells. In one embodiment, the neutralizing agents are capable ofcounteracting undesired concerted burst of cytokine expression/activityand, thus, are useful for the prevention, amelioration and treatment ofCRS associated with CART19 therapy.

In one embodiment, the treatment of CRS is performed around day 10-12post-infusion of CART19 cells.

In one embodiment, the second-line of therapy comprises administering asteroid to the patient. In another embodiment, the second-line oftherapy comprises administering one of more of a steroid, an inhibitorof TNFα, and an inhibitor of IL-6. An example of a TNFα inhibitor isentanercept. An example of an IL-6 inhibitor is Tocilizumab (toc).

EXPERIMENTAL EXAMPLES

The invention is further described in detail by reference to thefollowing experimental examples. These examples are provided forpurposes of illustration only, and are not intended to be limitingunless otherwise specified. Thus, the invention should in no way beconstrued as being limited to the following examples, but rather, shouldbe construed to encompass any and all variations which become evident asa result of the teaching provided herein.

Without further description, it is believed that one of ordinary skillin the art can, using the preceding description and the followingillustrative examples, make and utilize the compounds of the presentinvention and practice the claimed methods. The following workingexamples therefore, specifically point out the preferred embodiments ofthe present invention, and are not to be construed as limiting in anyway the remainder of the disclosure.

Example 1: Cytokine Therapy in Combination with CAR T Cell Infusion

The results presented herein demonstrate that patients followinginfusion of CAR T cells exhibit differential expression levels ofvarious cytokines. In some instances, the elevated levels of somecytokines are a result of the toxicity of the infused CAR T cells (FIG.1). It was observed that tocilizumab (anti-IL6) can ameliorate thetoxicity of CARs and seemingly preserve antitumor effects in 2 of 2patients (FIG. 2). Without wishing to be bound by any particular theory,it is believed that anakinra and other reagents that block IL-1 may alsobe useful in this regard. The data presented herein also demonstratesthat IL-1 is elevated in patients, and this may lead to the later risein IL-6. Anakinra is an IL-1Ra recombinant protein which binds to theIL1 receptors and blocks both IL-1 alpha and beta signaling. Anakinrahas a short ½ life. There is an advantage to use Anakinra to starttreating patients since both IL-1 alpha and beta would be blocked, andalso relieve the cytokine storm and keep the anti-tumor effect.

It was also observed that antibody interventions did not impart CART19cellular functionality as measured by Perforin and IFN-γ (FIG. 3).

Example 2: CD19-Redirected Chimeric Antigen Receptor T (CART19) CellsInduce a Cytokine Release Syndrome (CRS) and Induction of TreatableMacrophage Activation Syndrome (MAS) that can be Managed by the IL-6Antagonist Tocilizumab (toc)

Infusion of CART19 cells results in 100 to 100,000× in vivoproliferation, tumor lysis syndrome followed by durable antitumoractivity, and prolonged persistence in patients with B cell tumors. Theresults presented herein demonstrate that in vivo proliferation ofCART19 cells and potent anti-tumor activity therefrom is associated withCRS, leading to hemophagocytic lymphohistiocytosis (HLH), also termedMAS. Without wishing to be bound by any particular theory, it isbelieved that MAS/HLH is a unique biomarker that is associated with andmay be required for potent anti-tumor activity.

Autologous T cells were lentivirally transduced with a CAR composed ofanti-CD19 scFv/4-1BB/CD3-zeta, activated/expanded ex-vivo withanti-CD3/anti-CD28 beads, and then infused into ALL or CLL patients withpersistent disease after 2-8 prior treatments. CART19 anti ALL activitywas also modeled in a xenograft mouse model with high level of humanALL/human T cell engraftment and simultaneous detection of CAR T cellsand ALL using 2-color bioluminescent imaging.

The results presented herein provides updated results of 10 patients whoreceived CART19 cells, including 9 patients with CLL and 1 pediatricpatient with relapsed refractory ALL. 6/9 evaluable patient s had acomplete recovery (CR) or partial recovery (PR), including 4 sustainedCRs. While there was no acute infusional toxicity, all respondingpatients also developed CRS. All had high fevers, as well as grade 3 or4 hypotension/hypoxia. CRS preceded peak blood expression of CART19cells, and then increased in intensity until the CART19 cell peak(D10-31 after infusion). The ALL patient experienced the mostsignificant toxicity, with grade 4 hypotension and respiratory failure.Steroid therapy on D6 resulted in no improvement. On D9, noting highlevels of TNFα and IL-6 (peak increases above baseline: IFNγ at 6040x;IL-6 at 988x; IL-2R at 56x, IL-2 at 163x and TNFα at 17x), TNFα and IL-6antagonists (entanercept and toc) were administered. This resulted inresolution of fever and hypotension within 12 hr and a rapid wean fromventilator support to room air. These interventions had no apparentimpact on CART19 cell expansion or efficacy: peak of CART cells (2539CAR+ cells/uL; 77% of CD3 cells by flow) occurred on D11, and D23 bonemarrow showed CR with negative minimal residual disease (MRD), comparedto her initial on-study marrow which showed 65% blasts. Although she hadno history of CNS ALL, spinal fluid showed detectable CART19 cells (21lymphs/mcL; 78% CAR+). At 4 mo post infusion, this patient remained inCR, with 17 CART19 cells/uL in the blood and 31% CAR+CD3 cells in themarrow.

Clinical assessment of subsequent responding patients shows all hadevidence of MAS/HLH including dramatic elevations of ferritin andhistologic evidence of HLH. Peak ferritin levels range from 44,000 to605,000, preceding and continuing with peak T cell proliferation. Otherconsistent findings include rapid onset hepatosplenomegaly unrelated todisease and moderate DIC.

Subsequently, 3 CLL patients have also been treated with toc, also withprompt and striking resolution of high fevers, hypotension and hypoxia.One patient received toc on D10 and achieved a CR accompanied by CART19expansion. Another patient had rapid resolution of CRS following tocadministration on day 9 and follow up for response is too short. A 3rdCLL patient received toc on D3 for early fevers and had no CART-19proliferation and no response.

To model the timing of cytokine blockade, xenografts usingbioluminescent primary pediatric ALL were established and then treatedwith extra cells from the clinical manufacture. The CART19 cellsproliferated and resulted in prolonged survival. Cytokine blockade priorto T cell infusion with toc and/or etanercept abrogated disease controlwith less in vivo proliferation of infused CART19 cells, confirming theresult seen in the one patient given early toc (D3).

CART19 T cells can produce massive in-vivo expansion, long-termpersistence, and anti-tumor efficacy, but can also induce significantCRS with features suggestive of MAS/HLH that responds rapidly tocytokine blockade. Given prior to initiation of significant CART19proliferation, blockade of TNFα and/or IL-6 may interfere withproliferation and effector function, but if given at a point where cellproliferation is underway, toc may ameliorate the symptoms that havebeen observed that correlate with robust clinical responses.

Example 3 Remission of ALL by Chimeric Antigen Receptor-Expressing TCells

The results presented herein demonstrate that CAR T cells have clinicalactivity in acute lymphocytic leukemia (ALL). Briefly, two pediatricpatients with relapsed and refractory pre-B cell ALL were treated with10⁶ to 10⁷/kg T cell transduced with anti-CD19 antibody and a T-cellsignaling molecule (CTL019 CAR T cells; also referred to as CART19). TheCTL019 T cells expanded more than 1000-fold in both patients, andtrafficked to bone marrow. In addition, the CAR T cells were able tocross the blood brain barrier and persisted at high levels for at least6 months, as measured in the cerebral spinal fluid. Eight severe adverseevents were noted. Both patients developed a cytokine release syndrome(CRS) and B cell aplasia. In one child, the CRS was severe and cytokineblockade with etanercept and tocilizumab was effective in reversing thesyndrome, and yet did not prevent CAR T cell expansion and anti-leukemicefficacy. Complete remission was observed in both patients, and isongoing in one patient at 9 months after treatment. The other patientrelapsed with blast cells that no longer express CD19 approximately 2months after treatment.

The results presented herein demonstrate that CAR modified T cells arecapable of killing even aggressive treatment refractory acute leukemiacells in vivo. The emergence of tumor cells that no longer express thetarget indicates a need to target other molecules in addition to CD19 insome patients with ALL.

The in vivo expansion and robust anti-leukemic effects of CTL019(CART19) cells in 3 patients with CLL as been reported (Porter et al.,2011, N Engl J Med 365:725-33; Kalos et al., 2011, Science TranslationalMedicine 3:95ra73). CTL019 is a CAR that includes a CD137 (4-1BB)signaling domain and is expressed using lentiviral vector technology(Milone et al., 1009, Mol Ther 17:1453-64). The results presented hereindemonstrate the use of CTL019 in 2 pediatric patients with refractoryand relapsed ALL. Both patients had remission of leukemia, accompaniedby robust expansion of CTL019 in vivo with trafficking to marrow and theCNS. The anti-leukemic effects were potent since one patient hadchemotherapy refractory disease precluding allogeneic donor stem celltransplantation and the other patient relapsed after allogeneic cordblood transplantation and was resistant to blinatumomab (chimericbispecific anti-CD3 and anti-CD19) therapy.

The materials and methods employed in these experiments are nowdescribed.

Materials and Methods

CART19

CTL019 (CART19) production has been previously reported (Porter et al.,2011, N Engl J Med 365:725-33; Kalos et al., 2011, Science TranslationalMedicine 3:95ra73). CTL019 was detected and quantified in patientspecimens as previously reported (Porter et al., 2011, N Engl J Med365:725-33; Kalos et al., 2011, Science Translational Medicine3:95ra73).

Sample Draws and Processing

Samples (peripheral blood, bone marrow) were collected in lavender top(K2EDTA,) or red top (no additive) vacutainer tubes (Becton Dickinson).Lavender top tubes were delivered to the laboratory within 2 hours ofdraw, or shipped overnight at room temperature in insulated containersessentially as described (Olson et al., 2011, J Transl Med 9:26) priorto processing. Samples were processed within 30 minutes of receiptaccording to established laboratory SOP. Peripheral blood and marrowmononuclear cells were purified, processed, and stored in liquidnitrogen as described (Kalos et al., 2011, Science TranslationalMedicine 3:95ra73). Red top tubes were processed within 2 hours of drawincluding coagulation time; serum isolated by centrifugation, aliquotedin single use 100 μL aliquots and stored at −80° C. CSF was delivered tothe laboratory within 30 minutes of aspiration and cells in CSF werecollected by centrifugation of CSF fluid and processed for DNA and flowcytometry.

Q-PCR Analysis

Whole-blood or marrow samples were collected in lavender top (K2EDTA) BDvacutainer tubes (Becton Dickinson). Genomic DNA was isolated directlyfrom whole-blood and Q-PCR analysis on genomic DNA samples was performedin bulk using ABI Taqman technology and a validated assay to detect theintegrated CD19 CAR transgene sequence as described (Kalos et al., 2011,Science Translational Medicine 3:95ra73) using 200 ng genomic DNA pertime-point for peripheral blood and marrow samples, and 18-21.7 nggenomic DNA per time-point for CSF samples. To determine copy number perunit DNA, an 8-point standard curve was generated consisting of 5 to 10⁶copies CTL019 lentivirus plasmid spiked into 100 ng non-transducedcontrol genomic DNA. Each data-point (sample, standard curve) wasevaluated in triplicate with a positive Ct value in 3/3 replicates with% CV less than 0.95% for all quantifiable values. A parallelamplification reaction to control for the quality of interrogated DNAwas performed using 20 ng input genomic DNA from peripheral blood andmarrow (2-4.3 ng for CSF samples), and a primer/probe combinationspecific for non-transcribed genomic sequence upstream of the CDKN1Agene as described (Kalos et al., 2011, Science Translational Medicine3:95ra73). These amplification reactions generated a correction factor(CF) to correct for calculated versus actual DNA input. Copies oftransgene per microgram DNA were calculated according to the formula:copies calculated from CTL019 standard curve per input DNA (ng)×CF×1000ng. Accuracy of this assay was determined by the ability to quantifymarking of the infused cell product by Q-PCR. These blindeddeterminations generated Q-PCR and flow marking values of 11.1% and11.6%, respectively, for the CHOP-100 and 20.0% and 14.4%, respectively,marking for the CHOP-101 infusion products.

Soluble Factor Analysis

Whole blood was collected in red top (no additive) BD vacutainer tubes(Becton Dickinson), processed to obtain serum using establishedlaboratory SOP, aliquoted for single use and stored at −80° C.Quantification of soluble cytokine factors was performed using Luminexbead array technology and kits purchased from Life technologies(Invitrogen). Assays were performed as per the manufacturer protocolwith a 9 point standard curve generated using a 3-fold dilution series.The 2 external standard points were evaluated in duplicate and the 5internal standards in singlicate; all samples were evaluated induplicate at 1:2 dilution; calculated % CV for the duplicate measureswere less than 15%. Data were acquired on a FlexMAP-3D by percent andanalyzed using XPonent 4.0 software and 5-parameter logistic regressionanalysis. Standard curve quantification ranges were determined by the80-120% (observed/expected value) range. Reported values included thosewithin the standard curve range and those calculated by the logisticregression analysis.

Antibody Reagents

The following antibodies were used for these studies: MDA-CAR (Jena andCooper, 2013, L. Anti-idiotype antibody for CD19. PlosONE 2013; inpress), a murine antibody to CD19 CAR conjugated to Alexa647. Antibodiesfor multi-parametric immunophenotyping: T cell detection panels:anti-CD3-FITC, anti-CD8-PE, anti-CD14-PE-Cy7, anti-CD16-PE-Cy7,anti-CD19-PE-Cy7 anti-CD16-PE-Cy7. B cell detection panels:anti-CD20-FITC, anti-CD45-PE, anti-CD45-APC, anti-CD19-PE-Cy7,anti-CD19-PE, anti-CD34-PCP-e710 and anti CD34-APC were procured frome-Biosciences.

Multi-Parameter Flow Cytometry

Cells were evaluated by flow cytometry directly after Ficoll-Paqueprocessing, with the exception of the CHOP-101 baseline sample which wasevaluated immediately after thaw of a cryopreserved sample.Multi-parametric immunophenotyping for peripheral blood and marrowsamples was performed using approximately 0.2-0.5×10⁶ total cells percondition (depending on cell yield in samples), and for CSF samplesusing trace amounts of cells collected following centrifugation of CSFfluid, and using fluorescence minus one (FMO) stains as described in thetext. Cells were stained in 100 μL PBS for 30 minutes on ice usingantibody and reagent concentrations recommended by the manufacturer,washed, and resuspended in 0.5% paraformaldehyde and acquired using anAccuri C6 cytometer equipped with a Blue (488) and Red (633 nm) laser.Accuri files were exported in FCS file format and analyzed using FlowJosoftware (Version 9.5.3, Treestar). Compensation values were establishedusing single antibody stains and BD compensation beads (BectonDickinson) and were calculated by the software. The gating strategy forT cells was as follows: Live cells (FSC/SSC)>dump channel(CD14+CD16+CD19-PECy7) vs CD3+>CD3+. The general gating strategy for Bcells was as follows: Live cells (FSC/SSC)>SSC low events>CD19+. Moregating details for the CHOP-100 and CHOP-101 samples are described inthe individual Figures.

Molecular MRD Analysis

Molecular MRD analysis was performed by Adaptive Biotechnologies(Seattle, Wash.) and high-throughput next-generation sequencing of theBCR IGH CDR3 region using the Illumina HiSeq/MiSeq platform-basedimmunoSEQ assay (Larimore et al., 2012, J Immunol 189:3221-30). Forthese analyses, 701-6,000 ng (approximately 111,000-950,000 genomeequivalents) of genomic DNA isolated from whole blood or marrow samplesobtained from patients were subjected to combined multiplex PCR andsequencing followed by algorithmic analyses to quantify individual IGHCDR3 sequences in samples. Parallel amplifications and sequencing of theTCRB CDR3 region (Robins et al., 2009, Blood 114:4099-107) in eachsample were done to assess quality of DNA samples. For each patient, theIGH CDR3 nucleotide sequences assayed from samples of different timepoints were aligned using EMBL-EBI multiple sequence alignment tool(Goujon et al., 2010, Nucleic Acids Res 38:W695-9; Sievers et al., 2011,Mol Syst Biol 7:539). The dominant clone from the earliest time-pointsample was bioinformatically tracked across the assayed IGH CDR3sequences in the following time-point samples to identify presence ofsequences with 95% or greater pair-wise sequence identity. The totalsequencing reads for those sequences similar to the dominant clone arereported for each time-point.

The results of the experiments are now described.

Case Reports

CHOP-100 was a 7 yo girl in her second recurrence of ALL. She wasdiagnosed 2 years prior and achieved a minimal residual disease (MRD)negative remission, relapsing 17 months after diagnosis. She re-enteredremission after reinduction chemotherapy but recurred 4 months later,after which she did not respond tofurtheclofaribine/etoposide/cyclophosphamide. Her karyotype at baselinewas 48, XX, del(9)(p21.3), +11, del(14)(q2?q24), +16/46, XX[4].Peripheral blood mononuclear cells (PBMC) were collected by apheresisbefore the intensive chemotherapy, anticipating that there may beinsufficient circulating T cells available for cell manufacturing aftersuch intensive treatment. This patient was infused with CTL019 cellsthat had been anti-CD3/CD28 expanded and lentivirally transduced toexpress the anti-CD19 CAR in a total dose of 3.8×10⁸ cells/kg (1.2×10⁷CTL019 cells/kg) given over 3 consecutive days as previously described(Porter et al., 2011, N Engl J Med 365:725-33; Kalos et al., 2011,Science Translational Medicine 3:95ra73). She did not receivelymphodepleting chemotherapy before her CTL019 infusions, with the mostrecent cytotoxic therapy given 6 weeks before CTL019 infusion. Noimmediate infusional toxicities were noted, but she was hospitalized forlow-grade fevers which progressed to high fevers by day 4, and on day 5the patient was transferred to the pediatric ICU (CHOP-100, FIG. 4A).This was followed by rapid progression to significant respiratory andcardiovascular compromise requiring mechanical ventilation and bloodpressure support.

The second ALL patient was a 10 yo girl (CHOP-101) who had experiencedher second relapse after a 4/6 matched unrelated umbilical cordtransplant 28 months after diagnosis and 10 months before CTL019infusion. She had experienced graft vs. host disease (GVHD) after hertransplant, which resolved with treatment; she was off immunosuppressionat the time of her relapse. She did not subsequently re-enter remissionin spite of multiple cytotoxic and biologic therapies. Her baselinekaryotype was 46 XX, del(1)(p13), t(2;9)(q?21;q?21), t(3;17)(p24;q23),del(6)(q16q21), del(9)(q13q22), der(16)t(1;?;16)(p13;?p13.3)[9], //46,Xy[1]. Before PBMC collection, she was treated with two cycles ofblinatumomab (Bargou et al., 2008, Science 321:974-7) with no response.Her peripheral blood cells were 68% donor origin at the time of PBMCcollection. CTL019 T cells were manufactured and infused as a total doseof 10⁷ cells/kg (1.4×10⁶ CTL019 cells/kg) in a single dose, afteretoposide/cyclophosphamide chemotherapy given for lymphodepletion theweek before. Her bone marrow on the day before CTL019 infusion wasreplaced by a population of CD19+/CD34+ ALL cells, with variableexpression of CD19 by standard clinical flow cytometry (FIG. 7). She hadno immediate infusional toxicities, but developed a fever on D+6 and wasadmitted to the hospital. She experienced no cardiopulmonary toxicities,and did not receive glucocorticoids or anti-cytokine therapy. CHOP-101experienced fever of unknown origin, suspected to be due to cytokinerelease (FIG. 4B), myalgias and two days of confusion (grade 3), whichspontaneously resolved. She had no evidence of GVHD after the infusionof the CTL019 cells. Though these cells had been collected from thepatient, they were largely of donor (cord blood) origin.

Induction of Remission in Both Subjects

Both subjects had an increase in circulating lymphocytes and neutrophilsin the 2 weeks following CTL019 infusion, as shown by plots depictingtotal WBC, ALC, and ANC relative to timing of CTL019 infusion (FIG. 4C).Most of the lymphocytes were comprised of T cells that expressed thechimeric antigen receptor (FIG. 8), shown in more detail in FIG. 5. Inboth subjects, high-grade non-infectious fevers were documented,followed by elevations of LDH (FIG. 4A). The elevations of LDH and highgrade fevers were similar to those previously described in CLL patientsafter CTL019 infusion (Porter et al., 2011, N Engl J Med 365:725-33;Kalos et al., 2011, Science Translational Medicine 3:95ra73).Approximately one month after infusion, MRD negative (<0.01%)morphologic remission of leukemia was achieved in both subjects (Table1).

The clinical remission in CHOP-100 was associated with a deep molecularremission that has persisted for at least 9 months as of January 2013(Table 1). High-throughput DNA sequencing of the IGH locus revealed apronounced decrease in total IGH reads at D+23 in the blood and marrowof CHOP-100. The malignant clone was not detected in the blood or marrowin more than 1 million cell equivalents that were sequenced at D+180. Incontrast, T-cell receptor sequences were readily detected in blood andmarrow, indicating the integrity of the DNA tested at all timepoints.

TABLE 1 Induction of molecular remission in blood and bone marrow ofCHOP-100 and 101 Number of Tumor input Total IGH clone Timepoint genomes(cell Total TCRβ Total IGH unique Dominant frequency Patient Tissue(day) equivalents) reads reads reads clone reads (%) CHP959-100 Blood −1111,340 525,717 189 6 185 97.88 23 218,210 1,651,129 0 0 0 0.00 87288,152 1,416,378 0 0 0 0.00 180 420,571 1,276,098 6 2 0 0.00 Marrow −1317,460 348,687 59,791 318 59,774 99.97 23 362,819 1,712,507 37 2 3389.19 87 645,333 425,128 10 1 10 100.00 180 952,381 800,670 45 7 0 0.00CHP959-101 Blood −1 152,584 1,873,116 38,170 52 30,425 79.71 23 417,3711,462,911 92 5 18 19.60 Marrow −1 158,730 2,417,992 66,368 65 50,88774.43 23 305,067 1,978,600 1,414 11 946 66.90 60 916,571 N/A 530,833 206363,736 68.90Molecular analysis of minimal residual disease was performed on DNAisolated from whole blood or marrow

Toxicity of CTL019

Grade 3 and 4 adverse events are summarized in Table 2. Acute toxicitywas observed in both patients that consisted of fever, and a cytokinerelease syndrome (CRS) that evolved into a macrophage activationsyndrome (MAS). Both patients were monitored and given prophylaxis fortumor lysis syndrome. Both experienced substantial elevations of LDH,the causes of which were likely multifactorial but could have includedtumor lysis syndrome. Each uric acid value in CHOP-100 was either belownormal or in the normal range, and she received allopurinol only on days5-6. CHOP-101 received prophylactic allopurinol on days 0-14 and hadabnormal uric acid values of 4.8-5.7 on days 8-10, consistent with mildtumor lysis syndrome.

TABLE 2 Adverse events (grade 3 and 4) in CHOP-100 and CHOP-101 AE AECategory AE Toxicity Grade AE Description Duration CHP959-100 INFECTIONFebrile 3 Febrile neutropenia. 7 days neutropenia Temperature: Peaktemperature 40.7° C., resolved day 7 after administration oftocilizumab. CARDIAC GENERAL Hypotension 4 Shock requiring pressor 4days at support. Off all pressor grade 4, off support on day 7 other allpressors than weaning by day 12 dobutamine. VASCULAR Acute vascular 4Live-threatening see above leak syndrome pressor support or ventilatorysupport indicated PULMONARY/UPPER Adult Respiratory 4 Present,intubation 12 days RESPIRATORY Distress Syndrome indicated. Chest X-ray(ARDS) cleared on day 8. CHP959-101 INFECTION Febrile 3 Febrileneutropenia. 6 days neutropenia Peak temperature 40.3° C., resolved day6 NEUROLOGY Encephalopathy 3 Parents reported 3 days confusion. MRI wasnormal METABOLIC/ elevated AST 4 Peak AST Value 1060 1 day at LABORATORY(Grade 4) grade 4 METABOLIC elevated ALT 4 Peak ALT Value 748 1 day atLABORATORY (Grade 4) grade 4Adverse events were graded according to Common Terminology Criteria forAdverse Events 3.0

In CHOP-100, glucocorticoids were administered on D+5 with a briefresponse in the fever curve but without remission of hypotension. Asingle course of anti-cytokine therapy consisting of etanercept andtocilizumab was given on D+8 and was followed by rapid clinical effects:within hours she defervesced, was weaned off vasoactive medications andventilatory support as her clinical and radiologic ARDS resolved. Shedid not have laboratory evidence of a tumor lysis syndrome; however,biochemical evidence of MAS was noted with elevation of ferritin to45,529 ng/dl on D+11, coagulopathy with elevated d-dimer andhypofibrinogenemia, hepatosplenomegaly, elevation of transaminases,elevated LDH (FIG. 4C), and elevated triglycerides, as well as acytokine profile consistent with MAS. Her ferritin decreased to 2,368 byD+26 and the clinical and laboratory abnormalities of MAS resolved.

In CHOP-101, although there was no direct evidence of a tumor lysissyndrome other than fever and changes in LDH (FIG. 4C), she alsodeveloped features of MAS with elevations in ferritin to 33,360 on D+7,peaking at 74,899 on day 11, transaminases that reached grade 4 for 1day, and an elevated d-dimer in serum. These biochemical changes werereversible, as transaminases improved to grade 1 and the ferritindecreased to 3,894 by D+21. She was discharged from the hospital on dayD+16.

Both subjects developed prominent elevations in a number of cytokinesand cytokine receptors in the serum (FIG. 1B). In both patients,elevations in interferon-γ and IL-6 were most prominent. Theseobservations are similar to the pattern observed previously in patientswith CLL who also experienced remission of leukemia after CTL019infusion (Kalos et al., 2011, Science Translational Medicine 3:95ra73).The peak cytokine elevations were temporally correlated with systemicinflammation as judged by changes in core body temperature (FIG. 4C).

In Vivo Expansion of CTL019 in Patients with ALL

The fraction of CTL019 T cells in circulation progressively increased invivo to 72% (CHOP-100) and 34% (CHOP-101) of T cells (FIG. 5A). Theinitial transduction efficiency was 11.6% and 14.4% in the T cellsinfused in CHOP-100 and -101, respectively. Given that the total ALCincreased substantially in both patients (FIG. 4C), and that thefrequency of CTL019 cells progressively increased in vivo from thebaseline frequency (FIG. 8), there was a robust and selective expansionof CTL019 cells in both patients. The selective increase in T cellsexpressing CTL019 in both patients is consistent with an anti-leukemicmechanism involving CD19-driven expansion, and with the subsequentelimination of cells that express CD19 in both patients (FIG. 6 and FIG.9).

Molecular deep sequence analysis of TCRs in the peripheral blood andmarrow samples in CHOP-100 obtained at D+23, when >65% of CD3+ cells inperipheral blood and marrow were shown to be CTL019+ by flow cytometry,revealed the absence of a dominant T cell TCR clonotype in eithercompartment, with the 10 most abundant T cells present at frequenciesbetween 0.18-0.7% in bone marrow and 0.19 to 0.8% in peripheral blood.Six of the 10 dominant clones were shared between the two compartments.In addition both CD4 and CD8 CAR T cells are present. Thus, the CARTcells appear to proliferate after CD19-stimulated expansion, and not byTCR signals or clone-specific events such as activation by integrationof the lentivirus.

Trafficking and Morphology of CTL019 CART Cells in Marrow and CNS

CTL019 cells expanded more than 1000-fold in the peripheral blood andbone marrow (FIG. 5). The frequency of CTL019 cells increased to morethan 10% of circulating T cells by D+20 in both subjects (FIG. 8), withthe absolute magnitude of CTL019 expansion similar to that observed inpatients with CLL (Kalos et al., 2011, Science Translational Medicine3:95ra73). Unexpectedly, cells in the CSF also showed a high degree ofCTL019 gene marking and also persisted at high frequency out to 6 months(FIG. 5B). The trafficking of CTL019 cells to the CSF was surprisinggiven that neither patient had detectable CNS leukemia by cytospin atthe time of infusion or at the 1 month post-treatment evaluation.Furthermore, prior reports of CAR therapy for B cell malignancies havenot observed trafficking of CAR T cells to the CNS (Till et al., 2008,Blood 112:2261-71; Brentjens et al., 2011, Blood 118:4817-28; Savoldo etal., 2011, J Clin Invest 121:1822-5; Jensen et al., 2010, Biol BloodMarrow Transplant 16:1245-56; Till et al., 2012, Blood 119:3940-50;Kochenderfer et al., 2012, Blood 119:2709-20). The morphology of thelymphocytes in blood and CSF is shown for CHOP-100 and 101 in FIG. 5D.Since >70% of lymphocytes in circulation on D+10 were CTL019 cells(FIGS. 5A and 5B), most of the large granular lymphocytes shown in theleft panel of FIG. 5D are likely CTL019 cells. Similarly, since manylymphocytes in the CSF obtained from CHOP-101 on D+23 were CTL019 cells(FIGS. 5B and 5C), the cytospin of CSF lymphocytes in FIG. 5D mostlikely represents the morphology of CTL019 cells in vivo that havetrafficked to the CNS.

Induction of B Cell Aplasia

Both subjects had an elimination of CD19 positive cells in bone marrowand blood within 1 month after CTL019 infusion (FIG. 6, and FIG. 9). InCHOP-100, a large proportion of cells remaining in the marrow at D+6after infusion were CD19+CD20+ leukemic blast cells. This population ofcells was not detectable by D+23, an effect that is maintained beyond 9months in this patient (FIG. 9A). Given that CHOP-100 did not havechemotherapy in the 6 weeks preceding CTL019 infusion, this indicatesthat CTL019 cells were sufficient to ablate normal and leukemic B cellsin this case.

Emergence of CD19 Escape Variant in CHOP-101

CHOP-101 experienced a clinical relapse apparent in the peripheral bloodat 2 months after CTL019 infusion, as evidenced by the reappearance ofblast cells in the circulation. These cells were CD45dim positive, CD34positive and did not express CD19 (FIG. 6). The absence of the originaldominant CD34dim+CD34+CD19dim+ cells is consistent with a potentanti-leukemic selective pressure of the CTL019 CAR T cells directed toCD19 (FIG. 9B). Deep IGH sequencing revealed the presence of themalignant clone in peripheral blood and marrow as early as D+23 (Table1), despite a clinical assessment of MRD negativity by flow cytometry atthis timepoint (FIG. 7). In addition, deep sequencing of materialobtained at clinical relapse revealed that the CD45dimCD34+CD19− cellsare clonally related to the initial dominant CD45dim+CD34+CD19dim+cells, since they share the same IGH sequence.

Remission of ALL by Chimeric Antigen Receptor-Expressing T Cells

The results presented herein demonstrate the induction of remission ofrelapsed and refractory leukemia in the first two patients treated onthis protocol. Remission has been sustained in one subject and wasaccompanied by relapse due to the emergence of CD19 negative blasts inthe other subject. Genetically modified CTL019 cells trafficked to theCNS at high levels in both patients. Cytokine elevations were observedthat were on target, reversible, and temporally accompanied byelimination of blast cells that expressed CD19 in both subjects. Theinduction of complete remission in refractory CD19 positive ALLfollowing infusion of CAR T cells is encouraging, particularly given thelow frequency of remissions following the infusion of allogeneic donorlymphocyte infusions that do not express CARs (Kolb et al., 1995, Blood86:2041-50; Collins et al., 1997, J Clin Oncol 15:433-44; Collins etal., 2000, Bone Marrow Transplant 26(5):511-6). Deep sequencingtechnology indicated that the CTL019 CAR infusion was associated with asustained 5-log reduction in the frequency of malignant B cells inCHOP-100, further indicating potent antitumor effects inchemotherapy-refractory leukemia.

The unfortunate emergence of CD19-negative blast cells in one subject isconsistent with previous reports that document the existence ofCD19-negative precursor cells in some cases of ALL (Hotfilder et al.,2005, Cancer Research 65:1442-9; le Viseur et al., 2008, Cancer Cell14:47-58). It is possible that the coinfusion of CAR T cells redirectedto novel specificities in addition to CD19 might decrease the likelihoodof this event. Thus far, relapse with CD19-negative escape cells inadults with CLL after treatment with CTL019 cells have not been observed(Kalos et al., 2011, Science Translational Medicine 3:95ra73),suggesting that this issue may be specific for a subset of acuteleukemias. The induction of remission in CHOP-100 did not requireconcomitant chemotherapy, and is consistent with a previous reportshowing that remissions in CLL could be delayed for several weeksfollowing chemotherapy (Porter et al., 2011, N Engl J Med 365:725-33).Thus, concomitant administration of cytotoxic chemotherapy may not benecessary for CAR-mediated antitumor effects.

Both pediatric ALL patients experienced substantial toxicity afterCTL019 infusion. The induction of B-cell aplasia was observed, andindicates that the CART cells can function in the setting of relapsedacute leukemia. Both patients have also developed clinical andlaboratory evidence of cytokine release syndrome and macrophageactivation syndrome within a week of infusion. The cytokine profileobserved in these patients is similar to prior reports of cytokinepatterns in children with hemaphagocytosis and macrophage activationsyndrome or hemophagocytic lymphohistiocytosis (Tang et al., 2008, Br JHaematol 143:84-91; Behrens et al., 2011, J Clin Invest 121(6):2264-77).Macrophage activation syndrome is characterized by hyperinflammationwith prolonged fever, hepatosplenomegaly, and cytopenias. Laboratoryfindings characteristic of this syndrome are elevated ferritin,triglycerides, transaminases, bilirubin (mostly conjugated) and solubleinterleukin-2 receptor α-chain, and decreased fibrinogen (Janka et al.,2012, Annu Rev Med 63:233-46). Recent studies indicate that tocilizumab(anti-IL6) has promise for glucocorticoid resistant GVHD (Drobyski etal., 2011, Biol Blood Marrow Transplant 17(12):1862-8; Le Huu et al.,2012, J Invest Dermatol 132(12):2752-61; Tawara et al., 2011, ClinicalCancer Research 17:77-88), and the results presented herein areconsistent with these data.

The vigorous in vivo expansion of CTL019, persistent B-cell aplasia andprominent anti-leukemia activity imply substantial and sustainedeffector functions of the CTL019 cells in pediatric patients withadvanced ALL. The high efficiency of trafficking of CART cells to theCNS is encouraging as a mechanism for surveillance to prevent relapse ina sanctuary site such as the CNS (Pullen et al., 1993, J Clin Oncol11(5):839-49), and supports the testing of CAR T-cell-directed therapiesfor CNS lymphomas and primary CNS malignancies. With the exception ofB-cell aplasia, the duration of which is currently undefined, it isbelieved that the use of immune-based therapies such as CTL019 may havea favorable profile of long-term adverse effects compared to the highdoses of chemotherapy and radiation that are employed as the currentstandard of care for most cases of pediatric leukemia (Garcia-Manero andThomas, 2001, Hematol Oncol Clin North Am 15(1):163-205).

Induction of Complete Remissions of ALL by Chimeric AntigenReceptor-Expressing T Cells

Tocilizumab (anti-IL6) has promise for glucocorticoid resistant GVHD,and the results presented herein are consistent with these data.Further, it is interesting to note that in CHOP 100, the CRS manifestingas high fever, hypotension and multi-organ failure was resistant to thehigh doses of glucocorticoids administered over the previous 2 daysbefore cytokine directed therapy. Finally, in CHOP-100 the biphasicchanges in IL-1β, IL-1RA and IL-2 shown in FIG. 4B may have been relatedto cytokine-directed therapy with etanercept and tocilizumab.

The induction of remission in a patient refractory to blinatumomabtherapy further highlights the potency of CTL019 cells. The highefficiency of trafficking of CAR T cells to the CNS is encouraging as amechanism for surveillance to prevent relapse in a sanctuary site suchas the CNS, and supports the testing of CAR T-cell-directed therapiesfor CNS lymphomas and primary CNS malignancies. Neither patient hasexperienced cognitive effects that might be ascribed to the traffickingof T cells to the CNS.

Example 4: Summary Information

Various markers were measured in patients receiving CAR T cells. As anon-limiting example, Ferritin, Myoglobin, and plasminogen activatorinhibitor-1 (PAI-1) were measure; see FIGS. 10, 11 and 12, respectively.Elevated levels of these markers correlated with outcome. Patientsdesignated as -01, -03, -09, -100 and -101 were classified as completeresponders. Patients designated as -02, -05, -10 (second infusion andresponse around D70) and -12 were classified as partial responders.Patient designated as -06, -07 and -14 were classified asnon-responders.

The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated herein by reference intheir entirety. While this invention has been disclosed with referenceto specific embodiments, it is apparent that other embodiments andvariations of this invention may be devised by others skilled in the artwithout departing from the true spirit and scope of the invention. Theappended claims are intended to be construed to include all suchembodiments and equivalent variations.

1. A method of treating cancer in a patient, comprising administering tothe patient an effective amount of T cells genetically modified toexpress a chimeric antigen receptor (“CAR”) wherein the treatmentresults in cytokine release syndrome (“CRS”), and further comprisingadministering an IL-6 inhibitor to treat the CRS.
 2. The method of claim1, wherein the CAR comprises an extracellular domain having an antigenrecognition domain that targets a tumor antigen, a transmembrane domain,and a cytoplasmic domain.
 3. The method of claim 2, wherein the tumorantigen is selected from the group consisting of one or more of CD19,CD20, CD22, EGFRvIII, and IL3Ra.
 4. The method of claim 3 wherein thetumor antigen is CD19.
 5. The method of claim 4, wherein the antigenbinding moiety is fused with one or more intracellular domains selectedfrom the group consisting of a CD137 (4-1BB) signaling domain, a CD28signaling domain, a CD3zeta signal domain, and any combination thereof.6. The method of claim 1, wherein the CRS leads to hemophagocyticlymphohistiocytosis/macrophage activation syndrome.
 7. The method ofclaim 1 wherein the cancer is independently selected from the groupconsisting of: (i) a hematological malignancy selected from the groupconsisting of acute leukemia, acute lymphocytic leukemia, acutemyelocytic leukemia, acute myelogenous leukemia, myeloblastic leukemia,promyelocytic leukemia, myelomonocytic leukemia, monocytic leukemia,erythroleukemia, chronic leukemia, chronic myelocytic leukemia, chronicmyelogenous leukemia, chronic lymphocytic leukemia, polycythemia vera,lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma,Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplasticsyndrome, hairy cell leukemia and myelodysplasia; (ii) pre-B ALL(pediatric indication), adult ALL, mantle cell lymphoma, diffuse large Bcell lymphoma, further wherein the CAR is an anti-CD19 CAR; (iii) asolid tumor; (iv) a primary or metastatic cancer; and (v) a cancer thatis refractory or resistant to conventional chemotherapy.
 8. The methodof claim 1, wherein the CART cells are human T cells transduced in vitrowith a vector expressing the CAR, and the CAR T cells are autologous tothe patient.
 9. The method of claim 1, wherein the CART cells areadministered as a pharmaceutical composition in combination with one ormore pharmaceutically acceptable carriers, diluents or excipients. 10.The method of claim 1, wherein the IL-6 inhibitor is a nucleic acidinhibitor such as a small interfering RNA (siRNA) or antisense RNA, anantibody or a small chemical molecule.
 11. The method of claim 1,wherein the IL-6 inhibitor is an antibody.
 12. The method of claim 4,wherein the IL6-inhibitor is tocilizumab.
 13. The method of claim 12,wherein the cancer is selected from the group consisting of pre-B ALL(pediatric indication), adult ALL, mantle cell lymphoma and diffuselarge B cell lymphoma.
 14. The method of claim 12, wherein thetocilizumab is administered at a dose of 8 mg/kg.
 15. A method oftreating cancer in a patient comprising administering to the patient Tcells genetically modified to express a CAR wherein the treatmentresults in CRS, and further comprising administering an IL-6 inhibitorand a corticosteroid to treat the CRS.
 16. The method of claim 15,wherein the corticosteroid is methylprednisolone.
 17. The method ofclaim 15, wherein the IL-6 inhibitor is tocilizumab.
 18. The method ofclaim 15, wherein the IL-6 inhibitor is tocilizumab and thecorticosteroid is methylprednisolone.
 19. The method of claim 18,wherein the tocilizumab is administered at a dose of 8 mg/kg.
 20. Themethod of claim 19, wherein the CAR comprises an extracellular domainhaving an antigen recognition domain that targets a tumor antigen, atransmembrane domain, and a cytoplasmic domain.
 21. The method of claim20, wherein the tumor antigen is selected from one or more of CD19,CD20, CD22, EGFRvIII, and IL3Ra.
 22. The method of claim 21, wherein thetumor antigen is CD19.
 23. The method of claim 22, wherein the antigenbinding moiety is fused with one or more intracellular domains selectedfrom the group consisting of a CD137 (4-1BB) signaling domain, a CD28signaling domain, a CD3zeta signal domain, and any combination thereof.24. The method of claim 15, wherein the CRS leads to hemophagocyticlymphohistiocytosis/macrophage activation syndrome.
 25. The method ofclaim 15 wherein the cancer is independently selected from the groupconsisting of: (i) a hematological malignancy selected from the groupconsisting of acute leukemia, acute lymphocytic leukemia, acutemyelocytic leukemia, acute myelogenous leukemia myeloblastic leukemia,promyelocytic leukemia, myelomonocytic leukemia, monocytic leukemia,erythroleukemia, chronic leukemia, chronic myelocytic leukemia, chronicmyelogenous leukemia, chronic lymphocytic leukemia, polycythemia vera,lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma,Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplasticsyndrome, hairy cell leukemia and myelodysplasia; (ii) pre-B ALL(pediatric indication), adult ALL, mantle cell lymphoma, diffuse large Bcell lymphoma, further wherein the CAR is an anti-CD19 CAR; (iii) asolid tumor; (iv) a primary or metastatic cancer; and (v) a cancer thatis refractory or resistant to conventional chemotherapy.
 26. The methodof claim 15, wherein the CART cells are human T cells transduced invitro with a vector expressing the CAR, and the CAR T cells areautologous to the patient.
 27. The method of claim 15, wherein the CARTcells are administered as a pharmaceutical composition in combinationwith one or more pharmaceutically acceptable carriers, diluents orexcipients.
 28. The method of claim 15, wherein the IL-6 inhibitor is anucleic acid inhibitor such as a small interfering RNA (siRNA) orantisense RNA, an antibody or a small chemical molecule.